TAT介导EGFP在小鼠体内的分布及介导BDNF治疗PD模型鼠的实验研究
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摘要
帕金森病(Parkinson's Disease，PD)是中老年人常见的中枢神经系统慢性退行性疾病，是中老年人致残的主要原因之一。其发生机制是由于中脑黑质纹状体多巴胺(Dopamine，DA)能神经元的退行性变性，以及由此导致纹状体递质系统功能紊乱所致。其临床症状主要表现为静止性震颤、动作迟缓、运动减少、肌强直和姿势平衡障碍等而导致生活不能自理。PD的发病率及致残率很高，不仅给患者带来痛苦，也加重了社会和家庭的负担。目前对本病的临床治疗均属症状性治疗，不能阻多巴胺能神经元的进行性丢失，无法改变PD进行性发展的趋势。在胚胎干细胞移植治疗方面，因移植材料来源匮乏及移植细胞存活率低而受限制，基因治疗也面临着载体继发的安全性及毒性等问题。因此，寻找确切有效，易于临床应用的保护多巴胺能神经元的治疗措施，根本上预防PD的发生和阻止疾病的进展，已成为当前研究的热点。
脑源性神经营养因子(brain derived neurotrophic factor，BDNF)属神经营养素(neurotrophin)家族的成员，能够促进多种神经元的存活，尤其对多巴胺能神经元有很强的营养和保护作用。BDNF能促进多巴胺能神经元的存活、分化与生长，能特异性的保护多巴胺能神经元抵抗神经毒性物质的破坏作用，并参与PD的发病与病理进程，被认为是治疗PD的新一代药物，但因为BDNF为大分子物质，不能透过血脑屏障，脑室内用药又极其不方便，给临床的应用带来很大困难。TAT是HIV-1的反式激活蛋白，能够高效、快速的穿透磷脂双分子层的生物膜，不仅能跨膜转导进入细胞内部，而且还能够将与其共价结合的异源蛋白转导到组织及细胞内，甚至透过血脑屏障进入中枢神经系统。国外已有学者将TAT与相对分子质量为(15～200)×10~3的蛋白进行融合后，介导这些蛋白从细胞外到细胞内的直接跨膜转运。还有报道提到TAT与铜锌超氧化物歧化酶的融合蛋白跨膜进入胰岛β细胞可以达到治疗Ⅰ型糖尿病的目的。将TAT与P53和P27等肿瘤抑制蛋白输入含有腹膜肿瘤的小鼠模型，可以延长小鼠的寿命。TAT的转导作用具有速度快，不依赖于温度、能量、细胞膜抗体，而且对细胞没有损伤等特点，因此被认为是一种很有前途的运载工具。目前有关TAT融合蛋白的神经系统应用多集中在对脑缺血疾病的研究中，对于TAT融合脑源性神经营养因子治疗帕金森病的研究，国内外尚未见报道。
本课题首先利用增强绿色荧光蛋白(enhanced green fluorescent protein，EGFP)作为标记蛋白，构建TAT-EGFP重组载体并表达融和蛋白，静脉注射小鼠后观察融和蛋白进入体内各组织的情况，以证实TAT的蛋白转导能力，尤其是穿透血脑屏障进入脑组织的能力，为TAT携带药物治疗中枢神经系统疾病提供理论基础。随后构建TAT—BDNF重组载体并表达融和蛋白，经过在体外与多巴胺能神经元作用检测其生物学活性后，静脉注入PD大鼠模型，来观察TAT—BDNF对模型鼠的治疗作用，以期找到一种能静脉应用且能够穿透血脑屏障有效治疗PD的方法。
本研究分为以下三个部分：
第一部分TAT介导EGFP在小鼠体内的跨膜转运
方法：
1．以pEGFP-N3为模版，通过PCR及基因克隆技术，构建表达载体pEGFP。
2．人工合成编码TAT蛋白转导域的两条DNA片段，退火后得到双链。双链经过酶切及与pEGFP连接后，构建表达载体pTAT—EGFP。利用双酶切及基因测序来鉴定载体。
3．将重组载体转至大肠杆菌E．coli BL21(DE3)中，在IPTG的诱导下，优化表达条件，表达出融合蛋白TAT—EGFP。SDS—PAGE凝胶电泳检测蛋白的分子量。同时表达对照蛋白EGFP。
4．融和蛋白经过Ni离子亲和层析柱纯化。
5．将得到的纯化后的融合蛋白经尾静脉注入小鼠的体内，4h后取脑、心肌、肝、脾、肾等器官作冰冻切片，荧光显微镜下观察融合蛋白在组织中的分布。以尾静脉注射EGFP蛋白作为对照组。
结果
1．琼脂糖凝胶电泳检测pEGFP-N3的PCR产物为720bp，符合预期的大小。
2．重组质粒pEGFP与pTAT—EGFP双酶切后得到的共同产物为720bp的EGFP与5000bp的pET28a质粒，片段符合预期的大小。经过基因测序，与Genbank上对照，序列达到100％，表明EGFP基因正确的插入到载体上，成功得到重组质粒pTAT—EGFP及pEGFP。
3．在IPTG的诱导下，发现0.5mmol／L IPTG、30℃诱导3h时融合蛋白TAT—EGFP以可溶性表达最高，0.5mmol／L IPTG、30℃诱导4h时蛋白EGFP以可溶性表达最高。SDS—PAGE检测发现TAT—EGFP分子量约为28KD，EGFP的分子量约为27KD，符合预期的大小。
4．经过Ni离子亲和层析柱纯化后，得到纯度大于85％的蛋白。
5．小鼠尾静脉注射融合蛋白TAT—EGFP及对照EGFP蛋白，4h后TAT—EGFP组在脑、肝、肾、脾、心、骨骼肌等各个器官组织中均检测到绿色的荧光，其中以肝、脾、肾中最多，心肌、脑其次，骨骼肌中亦有分布，对照组小鼠组织切片未检测到荧光。
第二部分pTAT—BDNF载体构建、表达及TAT—BDNF对多巴胺能神经元的营养作用
方法：
1．提取人脑组织RNA，设计合成引物，RT—PCR的方法克隆人BDNF基因。
2．将酶切的BDNF基因与原核表达载体pTAT／HA相连接，构建重组表达载体pTAT／HA—BDNF。双酶切及基因测序鉴定重组质粒。
3．将质粒转至大肠杆菌E．coli BL21(DE3)中，在IPTG的诱导下，优化表达条件，表达出融合蛋白TAT—BDNF。SDS—PAGE凝胶电泳检测蛋白的分子量。
4．包涵体蛋白经过洗涤、变性溶解后过Ni离子亲和层析柱纯化。
5．Western-blot进一步检测融合蛋白TAT—BDNF的完整性。
6．取14d孕鼠，分离培养胎鼠的中脑神经元细胞，用TH及MAP2免疫荧光细胞化学染色鉴定多巴胺能神经元细胞。
7．原代培养多巴胺能神经元细胞，1w换2次液，观察细胞的生长状况。
8．原代培养至第6d，分成5组，第1、2组暂不加干预因素，第3、4、5组加入不同浓度的TAT—BDNF，至第10d，除第一组外，均加入100μmol／L的6～0HDA，继续培养24h后，通过MTT检测细胞的活力，来观察TAT—BDNF对多巴胺能神经元的保护作用。
9．各组细胞在加入6—OHDA 24h后，Hoechst33258／PI双重荧光染色检测细胞凋亡，观察TAT—BDNF对多巴胺能神经元的保护作用。
结果：
1．常规提取出人脑RNA，用巢式PCR的方法得到约763bp的人BDNE基因，与预期相符。
2．质粒pTAT／HA—BDNF用EcoRⅠ和XhoⅠ酶切鉴定，切出与BDNF的PCR产物大小相当的片段，以及与载体pTAT／HA(3.0kb)大小一致的片段，表明人BDNF基因已正确插入载体pTAT／HA。pTAT／HA—BDNF重组质粒测序结果与Gnebank登录的基因序列相比，序列完全相符，表明成功得到重组载体pTAT／HA—BDNF。
3．重组pTAT／HA—BDNF转化BL21(DE3)菌后，经IPTG诱导后产生一条约35KD的特异蛋白条带，与预期的蛋白质分子量大小相符。诱导条件优化后发现，在IPTG为0.5mmol，37℃，4h的条件下诱导的目标蛋白总蛋白比值最高。
4．蛋白几乎都是以包涵体的形式存在于沉淀中，包涵体经过洗涤，变性溶解后，经过镍离子亲和层析柱纯化得到较为纯净的蛋白。
5．Western—blot检测TAT—BDNF同时具有BDNF的免疫原性及多聚组氨酸的免疫原性，说明表达的融合蛋白完整。
6．TH及MAP2免疫荧光细胞化学染色鉴定多巴胺能神经元细胞，发现多巴胺能神经元细胞占80％以上，完全能达到实验的需要。
7．原代培养的不同天数观察细胞，可见到细胞贴壁，长出神经突起并延长交织呈网状。
8．四唑盐(MTT)比色法测定细胞生长指数，5、20、50μg／L TAT—BDNF预处理组多巴胺能神经元细胞活力较6—OHDA组比均有显著提高(P＜0.01)，而与正常对照组比较无明显差异，其中50、20μg／L TAT—BDNF较5μg／L TAT—BDNF细胞活力更高(P＜0.05)。
9．Hoechst33258／PI双重荧光染色可以看到，正常培养的多巴胺能神经元仅有少量的细胞凋亡，经6—OHDA作用后可见大量的细胞凋亡，经过TAT—BDNF预处理的细胞凋亡的情况较6—OHDA作用组明显减少，而50μg／ml、20μg／ml实验组较5μg／ml实验组效果明显。
第三部分融和蛋白TAT—BDNF对PD大鼠的治疗作用
方法：
1．用6—OHDA与立体定向仪，制作PD大鼠纹状体二点单侧毁损模型。
2．检测TAT—BDNF在脑内的分布：大鼠随即分为2组：实验组及对照组，每组3只，共6只。实验组在制作模型当时，尾静脉注射0.1ml(500μg)的TAT—BDNF，转导对照组尾静脉注射0.1ml的生理盐水，4h后取中脑组织，用HA免疫荧光组织化学染色来检测TAT—BDNF是否进入脑组织内。
3．提取实验组及对照组大鼠中脑组织，Western—blot检测TAT—BDNF在脑组织内的分布。
4．TAT—BDNF对PD大鼠的治疗作用：大鼠随即分为4组：正常组，模型组，实验组及对照组，每组16只，共64只。实验组在制作模型当时，尾静脉注射0.1ml(500μg)的TAT—BDNF，以后每7d注射一次，至28d。对照组尾静脉注射0.1ml的生理盐水，每7d注射一次，至28d。各组在不同的时间点作行为学检测，记录并比较阿扑吗啡诱发的旋转圈数。
5．第28d时，TH免疫组织荧光检测各组大鼠黑质中多巴胺能神经元，计算细胞核轮廓清晰的TH免疫阳性神经元，比较各组之间的差异。
6．第28d时，TUNEL法检测大鼠中脑黑质凋亡神经元，计算每张冠状切片的TUNEL阳性细胞数，比较各组之间的差异。
结果：
1．采用荧光免疫组织化学染色后在荧光显微镜下观察，静脉注射TAT—BDNF蛋白4h后PD大鼠的中脑内能看到均匀分布的由HA标记的融合蛋白，而对照组在荧光显微镜下不能看到荧光标记的TAT—BDNF，证实TAT能介导BDNF穿过血脑屏障进入中枢神经系统。
2．Western—blot分析结果显示在注射TAT—BDNF融合蛋白4h后的大鼠中脑的蛋白提取液中能检测到具有多聚组氨酸抗原特性的蛋白质。而从转导对照组大鼠中脑提取的蛋白质则不能与多聚组氨酸抗体起反应。说明融合蛋白已穿过血脑屏障分布到中枢神经组织内。
3．模型组和对照组大鼠旋转行为最为明显，实验组转速有所下降，旋转的幅度也减轻，表现为旋转的圆周扩大，而正常组无明确的旋转行为。在实验组治疗的前14d统计结果显示实验组和对照组之间没有差异，第21d时两组有显著性差异，第28d时两组比较有极显著差异。
4．正常组大鼠黑质内TH阳性神经元主要分布于黑质网状部，沿黑质区长轴方向排列，数量较多，胞体较大，呈锥体形或椭圆形，纤维细长，量多而密集神经元突起较明显。模型组TH阳性神经元明显减少，分布稀疏，神经元胞体轮廓及突起不清晰。TAT—BDNF实验组TH阳性神经元及阳性纤维较对照组明显改善，神经元及纤维数量明显增多。
5．TUNEL法显示模型组及对照组大鼠损毁侧中脑黑质区有大量染色阳性细胞，细胞核固缩，呈棕黄色，并可见裂解的棕色凋亡小体。通过计算机图像分析系统统计平均每张冠状切片的凋亡细胞数。结果显示损伤后注射TAT—BDNF蛋白的大鼠平均每张中脑黑质切片的TUNEL阳性细胞数明显小于对照组，正常组未见阳性细胞。说明6—OHDA注入纹状体后可逆向引发同侧黑质DA能神经元以凋亡形式发生退化，TAT—BDNF可能通过抗凋亡的作用保护大鼠多巴胺能神经元。
结论
1．小鼠尾静脉注射融合蛋白TAT—EGFP 4h后，在肝、脑、肾、脾、心肌及骨骼肌等组织内均有绿色荧光的分布，而注射了EGFP的对照组组织中未见荧光，提示了TAT能够携带EGFP穿透生物膜屏障进入各个组织，甚至是穿透血脑屏障进入中枢神经系统，为TAT携带大分子蛋白物质治疗中枢神经系统疾病提供了理论基础。
2．首次将表达的融和蛋白TAT—BDNF与多巴胺能神经元作用后，证实TAT—BDNF能保护细胞抵抗6—OHDA的神经毒性，细胞存活率明显提高。提示合成的TAT—BDNF具有与BDNF相同的神经保护的生物学活性。
3．采用纹状体二点单侧毁损术成功制备了PD大鼠模型，从大鼠旋转行为的变化可看出6—OHDA的对黑质内DA能神经元的毁损是渐进性的，比黑质毁损法制备的模型能更好地模拟PD的发病过程。提示该模型能更好地显示在PD早期DA能神经元进行性变性阶段药物的治疗作用。
4．首次将融和蛋白TAT—BDNF用于PD大鼠模型的治疗，实验组大鼠较对照组在行为学上有很大的改善，TH阳性神经元增多，TUNEL染色显示凋亡细胞减少。提示TAT能携带BDNF穿透血脑屏障进入中枢神经系统起到治疗作用，给临床外源大分子物质治疗PD提供了一条新思路。
Parkinson 's disease (PD) is a common chronic degenerative disease of central nervous system (CNS) in the middle—aged and old people. It is also one of the main causes of disability for these people. The pathology of this disease is retrograde degeneration of dopaminergic neuron in the substantia nigra of midbrain and striatum which result in the functional disorder of transmitter system in striatum system. Clinically, cardinal symptoms are static tremor, movement retardation, hypokinesia, rigidity and posture balance disturbance and so on. Incidence and disability of PD are high, which puts a heavy burden on society,family and individual.At present, only symptomatic treatment can not prevent dopaminergic neuron from degenerating. Transplantation of embryonic stem cells is limited because of insufficient materials source and the low survival rate of transplanted cells. Gene therapy is also faced with the problems of safety and toxicity. So it is very important to find some new agents and medicines to prevent and treat PD.
Brain derived neurotrophic factor(BDNF) belongs to the family of neurotrophin. It can promote many kinds of neurons to survive, especially the strong function of nutrition and protection to the dopaminergic neuron. BDNF can not only promote survival differentiation and growth of the dopaminergic neuron but also prevent dopaminergic neuron from degeneration caused by neurotoxicity substance.So BDNF could be a new kind of drug to treat Parkinson. But BDNF is macromolecular substance which can not permeate blood brain barrier(BBB), so it is difficult to apply clinically. TAT is trans—activator of HIV—1,and it can transmembrane into cell and transduct the covalent—bonding heterogenic protein into cell, At the same time, it also can permeate the BBB. Foreign scholars had fused TAT with protein whose molecular weight was (15～200)×10~3 and found these fusion proteins could been transducted into cells. It was reported that TAT fused SOD—Cu,Zn could transmembrane the beta Cell of the islet to treat diabetes. Also TAT fused the p53 and p27 can lengthen the life span of the mice which suffered from the tumor. At present, the study of TAT fusion protein in the field of nerves system focuses on cerebral ischemia. There is no report that the TAT fusion protein treats the PD.
We firstly utilized the enhanced green fluorescent protein(EGFP) as marker, constructed the plasmid TAT—EGFP and expressed the fusion protein, observing the protein distribution in different tissues after intravenous injecting the protein into mice, so that we can confirm the protein transduction ability, especially the ability of permeating BBB. Facts mentions aboveprovided the theory foundation of TAT mediates medicine to treat central nervous system disease. Then we constructed the plasmid TAT—BDNF and expressed the fusion protein, after detecting its neuroprotective activity by incubation with dopaminergic neuron in vitro. TAT—BDNF was intravenously injected into rat model of PD. Its neuroprotective effect was evaluated by observing the ethology and histomorphology of rats.in order to find a convenient and effective treatment for PD.
The Study Includes Three Parts:
PartⅠTAT Mediates EGFP to Transmembrane Transport into Mice Tissues
Methods
1. With the template pEGFP-N3, pEGFP was constructed by PCR and gene cloning technique.
2. Double strands was obtained by annealing the two synthesized DNA fragment encoding TAT protein transduction domain, then it was ligated to pEGFP after digestion by restriction enzyme to construct the expression vector of pTAT—EGFP.
3. pTAT—EGFP was transformed into E.coli BL21 (DE3), the fusion protein was expressed by IPTG. Molecular weight was detected by SDS—PAGE gel electrophoresis. Protein EGFP was obtained also in this step.
4. The protein was purified by Ni~(2+) affinity column.
5. The fusion protein was intravenously injected into mice by vena caudalis. Four hours later, frozen section was made from brain, heart muscle, liver, spleen, kidney and so on. Distribution of the fusion protein in different tissues was observed in fluorescence microscope.At the mean time, EGFP protein was intravenously injected into the control group mice.
Results
1. 720bp PCR product was confirmed by agarose gel electrophoresis, which was in line with the expectancy of EGFP.
2. The enzyme digestion products of recombinant plasmid pEGFPand pTAT—EGFP are 720bp and 5000bp, it is consistent with the expectancy of EGFP and pET28a. After gene sequencing, the recombinant plasmid sequence is 100% consistent with the sequence in Genebank. It showed that the EGFP gene was inserted the plasmid correctly, the recombinant plasmid were obtained successfully.
3. induced by IPTG, the expressed fusion protein TAT—EGFP was maximum in 0.5 mmol/L IPTG、30℃and 3h, in the same way, the expressed protein EGFP was maximum in 0.5 mmol/L IPTG, 30℃and 4h. Molecular weight of TAT—EGFP and EGFP are 28KD and 27KD by SDS-PAGE gel electrophoresis detection.
4. The purity coefficient of protein exceeds 85% after purification by Ni~(2+) affinity column.
5. Four hours later, in experimental group mice, green fluorescence was detected in different tissues, including brain, heart muscle, liver, spleen, kidney and so on. Among them, the fluorescence in liver and kidney were the most obvious, while in heart muscle and brain were moderate, and so did in skeletal muscle. In control group mice no fluorescence were detected in all tissues.
PartⅡExperiment Study of Construction and Expression of pTAT—BDNF and the Neuroprotective Effect of TAT—BDNF to Aminergic Neuron
Methods
1. RNA was extracted from human brain tissue, and human BDNF gene was clonedby RT—PCT.
2. BDNF gene was ligated to prokaryotic expression vector pTAT/HA afterdigestion by restriction enzyme,then the recombinant expression vector of pTAT/HA—BDNF was constructed.The recombinant vector was identified by double restriction enzyme digestion and gene sequencing.
3. pTAT/HA-BDNF was transformed into E.coli BL21 (DE3), the fusion protein TAT—BDNF was expressed by IPTG. Molecular weight was detected by SDS—PAGE gel electrophoresis.
4. Inclusion was purified by Ni~(2+) affinity column after washing and denaturation.
5. Inclusion body of the fusion protein TAT—BDNF was further detected by Western—blot.
6. Embryos were taken out from the pregnant mice, the midbrain neurons were isolated and cultivated. Dopaminergic neuron was identificated by TH and MAP2 double immunofluorescence stain.
7. Dopaminergic neurons were primary cultivated, and nutrient medium was exchanged twice a week. The growth state was observed by microscope.
8. Cells were divided into 5 groups when cultivated in the sixth day, and different concentration TAT—BDNF was added into the third, the forth and the fifth group. In the tenth day, except for the first group, 100μg 6—OHDA was added into all groups, then cells continue to been cultivated for 24 hours. Protection to dopaminergic neuron was observed by detecting cell vigor using MTT method.
9. Apoptosis was detected by hoechst 33258 and PI double immunofluorescence stain after 6—OHDA was added.
Results
1. Human BDNF gene of 763bp was obtained by nest PCR from the human brain,it was in line with the expectancy of BDNF.
2. The product of pTAT/HA—BDNF digestedby restriction enzyme was about 720bp and 3.0kb which is in line with the BDNF gene and the pTAT/HA vector, so it showed that human BDNF had already inserted the vector correctly. Gene sequencing confirmed that pTAT/HA—BDNF according with the sequence in Gene bank. The recombinant vector was obtained successfully.
3. pTAT/HA—BDNF was transformed into E.coli BL21 (DE3), and SDS—PAGE gel electrophoresis showed the protein of 35KD was produced by IPTG By changing the expression condition, maximum fusion protein was expressed.
4. Most of expressed protein was reside in the sediment in the style of occlusion body, occlusion body was purified by Ni~(2+) affinity column after washing and denaturation.
5. TAT—BDNF had the immunogenicity of both poly—histidine and BDNF by Western—blot, which showed that the fusion protein was integrity.
6. It was identified that dopaminergic neuron were above 80% in the midbrain neurons by TH and MAP2 double immunofluorescence stain. This satisfied us that it is enough for the next step.
7. The dopaminergic neuron was observed in different time, cell adherence,nervous process eruption and extension into reticulation can be observed by microscope.
8. Cell growth index was detected by MTT chromatometry, the result showed that cell survival rate in the group of pre—disposal treatment using different concentration TAT—BDNF increased significantly than that of 6—OHDA group.Futhermore, the cell survival rate of 50, 20μg BDNF is higher that of 5μg BDNF.
9.Slight apoptosis can be observed in normally cultivated dopaminergic neurons by Hoechst 33258 and PI double immunofluorescence stain,a lot of cell apoptosis in 6—OHDA group, apoptosis in the group of pre—disposal TAT—BDNF treatment decreased obviously than that of 6—OHDA group. Furthermore, neuroprotective effect of 50μg/ml and 20μg/ml is more significant than that of 5μg/ml treatment.
PartⅢThe Therapeutical Effect of Fusion TAT—BDNF on Rat Model of PD
Methods
1 Using stereotaxic apparatus, PD models were made by injecting the 6—OHDA into unilateral Striatum of the rats.
2 Distribution detection of the TAT—BDNF: Rats were randomly divided into two groups—study group and control group, and there are 3 rats in each group. Study group rats were intravenously injected 0.1ml(500μg) TAT—BDNF by vena caudalis when rats were made into the PD models, rats of control group were injected 0.1ml physiological saline. Four hours later, rats were killed and midbrain tissues were taken out, whether or not TAT—BDNF got into brain tissue was observed by HA immunofluorescence histochemical stain.
3 Midbrain tissues of rats were extracted and TAT—BDNF was identified by Western—blot so that we can confirm that TAT—BDNF distributed into brain tissues.
4 Neuroprotective effect of TAT—BDNF rats model of PD: Rats were randomly divided into four groups—normal group,model group, study group, control group. There were 16 rats in each group. Study group rats were intravenously injected 0.1ml(500μg) TAT—BDNF by vena caudalis when they were made into the PD models, then every 7day to 28 day, same dosage TAT—BDNF was injected into Study group rats. Control group rats were injected 0.1ml physiological saline. In different time, rats of every group were examined in ethology, and rotation circle numbers were record and compared.
5 Dopaminergic neurons that TH immunization was positive were counted by TH immunohistofluorescence in 28th day. Number of dopaminergic neurons in different group was compared.
6 Substantia nigra of midbrain was detected by TUNEL, and TUNNEI positive cells were counted and compared in the different groups.
Results
1 Using the HA immunofluorescence histochemical stain method, we saw the fusion protein homogeneous distribution in mid—brain of rat by fluorescence microscope, However, no fluorescence was detected in the rats of control group, which confirmed that TAT can mediate BDNF to permeate the BBB in CNS of rats.
2 Western-blot analysis showed that there was fusion protein in the midbrain of rats which was injected the TAT—BDNF,however, there was no fusion protein in the midbrain of control rats. It indicated that fusion protein had already permeated the BBB into central nervous system of rats.
3 The rotational behavior of study group rats was distinctly different from model group threats. The rotation speed of study group rats was decrease,and the extent of rotation was lighten. Rats of normal group did not rotate. Statistical results showed that the rotational behavior in four group rats was not discrepancy in first two weeks. However, in the third week, the rotational behavior of the study group rats was significantly different from the control group rats,and in the forth week, the difference was extreme in two group rats.
4 In normal rats, there were many large cells of TH—immunoreactive(IR) positive located mainly in the reticular compacta of substantia nigra, arranging along the long axis of substantia nigra region. These cells were pyramid or ellipse and their fibers were slender. TH positive cells were very few in rats of model group, cells distributed sparseness and their outline and neurite were fuzziness. TH positive cells and fibers were increased significantly in rats of study group, and the outline and neurite of neuros were distinct.
5 A great quantity of positive cells in substantia nigra of midbrain were observed in the rats of model group and the control group by TUNEL, these cell nucleuses were buffy and pyknosis, and brown apoptotic bodies were lytic. Analysis results showed that the positive cells in study group rats were less than that of control group significantly. There were not positive cells in normal group rats. It illustrated that the degeneration of DA neurons was related with apoptosis after 6—OHDA was injected into striatum of rats, and TAT—BDNF can keep DA neurons from apoptosis.
Conclusions:
1 Green fluorescence was detected in different tissues in study rats,such as brain, heart muscle, liver, spleen, kidney and so on, After injecting TAT—EGFP by vena caudalis while no fluorescence was detected in all tissues of control group mice, which indicated that TAT can mediated EGFP to permeat into tissues of mice, especially into CNS. These results provide us a new way to treat CNS disease using large molecular substance.
2 Fusion protein TAT—BDNF that we expressed could protect the dopaminergic neurons from the neurotoxicity of 6—OHDA, which indicated that TAT—BDNF has the neuroprotective activity.
3 We made the rats model of PD by two point injecting 6—OHDA into unilateral striatum of the rats successfully. The damage of 6—OHDA to dopaminergic neurons is gradually, so this model was more similar to the process of Parkinson disease than the models with other methods. Which indicated that this model is available for observing the therapeutic effect of medicine at the stage of progressive degeneration of neurons.
4 When fusion protein TAT—BDNF was used to treat the rats model of PD, we found that the ethology of study group rats was improved more obviously than the control group rats. There were more TH positive neurons and less TUNEL positive neurons in study group rats than the control group rats. These illustrated that TAT—BDNF could permeat BBB into the central nervous system, and TAT—BDNF had the neuroprotetive effect to dopaminergic neuron.
脑源性神经营养因子(brain derived neurotrophic factor，BDNF)属神经营养素(neurotrophin)家族的成员，能够促进多种神经元的存活，尤其对多巴胺能神经元有很强的营养和保护作用。BDNF能促进多巴胺能神经元的存活、分化与生长，能特异性的保护多巴胺能神经元抵抗神经毒性物质的破坏作用，并参与PD的发病与病理进程，被认为是治疗PD的新一代药物，但因为BDNF为大分子物质，不能透过血脑屏障，脑室内用药又极其不方便，给临床的应用带来很大困难。TAT是HIV-1的反式激活蛋白，能够高效、快速的穿透磷脂双分子层的生物膜，不仅能跨膜转导进入细胞内部，而且还能够将与其共价结合的异源蛋白转导到组织及细胞内，甚至透过血脑屏障进入中枢神经系统。国外已有学者将TAT与相对分子质量为(15～200)×10~3的蛋白进行融合后，介导这些蛋白从细胞外到细胞内的直接跨膜转运。还有报道提到TAT与铜锌超氧化物歧化酶的融合蛋白跨膜进入胰岛β细胞可以达到治疗Ⅰ型糖尿病的目的。将TAT与P53和P27等肿瘤抑制蛋白输入含有腹膜肿瘤的小鼠模型，可以延长小鼠的寿命。TAT的转导作用具有速度快，不依赖于温度、能量、细胞膜抗体，而且对细胞没有损伤等特点，因此被认为是一种很有前途的运载工具。目前有关TAT融合蛋白的神经系统应用多集中在对脑缺血疾病的研究中，对于TAT融合脑源性神经营养因子治疗帕金森病的研究，国内外尚未见报道。
本课题首先利用增强绿色荧光蛋白(enhanced green fluorescent protein，EGFP)作为标记蛋白，构建TAT-EGFP重组载体并表达融和蛋白，静脉注射小鼠后观察融和蛋白进入体内各组织的情况，以证实TAT的蛋白转导能力，尤其是穿透血脑屏障进入脑组织的能力，为TAT携带药物治疗中枢神经系统疾病提供理论基础。随后构建TAT—BDNF重组载体并表达融和蛋白，经过在体外与多巴胺能神经元作用检测其生物学活性后，静脉注入PD大鼠模型，来观察TAT—BDNF对模型鼠的治疗作用，以期找到一种能静脉应用且能够穿透血脑屏障有效治疗PD的方法。
本研究分为以下三个部分：
第一部分TAT介导EGFP在小鼠体内的跨膜转运
方法：
1．以pEGFP-N3为模版，通过PCR及基因克隆技术，构建表达载体pEGFP。
2．人工合成编码TAT蛋白转导域的两条DNA片段，退火后得到双链。双链经过酶切及与pEGFP连接后，构建表达载体pTAT—EGFP。利用双酶切及基因测序来鉴定载体。
3．将重组载体转至大肠杆菌E．coli BL21(DE3)中，在IPTG的诱导下，优化表达条件，表达出融合蛋白TAT—EGFP。SDS—PAGE凝胶电泳检测蛋白的分子量。同时表达对照蛋白EGFP。
4．融和蛋白经过Ni离子亲和层析柱纯化。
5．将得到的纯化后的融合蛋白经尾静脉注入小鼠的体内，4h后取脑、心肌、肝、脾、肾等器官作冰冻切片，荧光显微镜下观察融合蛋白在组织中的分布。以尾静脉注射EGFP蛋白作为对照组。
结果
1．琼脂糖凝胶电泳检测pEGFP-N3的PCR产物为720bp，符合预期的大小。
2．重组质粒pEGFP与pTAT—EGFP双酶切后得到的共同产物为720bp的EGFP与5000bp的pET28a质粒，片段符合预期的大小。经过基因测序，与Genbank上对照，序列达到100％，表明EGFP基因正确的插入到载体上，成功得到重组质粒pTAT—EGFP及pEGFP。
3．在IPTG的诱导下，发现0.5mmol／L IPTG、30℃诱导3h时融合蛋白TAT—EGFP以可溶性表达最高，0.5mmol／L IPTG、30℃诱导4h时蛋白EGFP以可溶性表达最高。SDS—PAGE检测发现TAT—EGFP分子量约为28KD，EGFP的分子量约为27KD，符合预期的大小。
4．经过Ni离子亲和层析柱纯化后，得到纯度大于85％的蛋白。
5．小鼠尾静脉注射融合蛋白TAT—EGFP及对照EGFP蛋白，4h后TAT—EGFP组在脑、肝、肾、脾、心、骨骼肌等各个器官组织中均检测到绿色的荧光，其中以肝、脾、肾中最多，心肌、脑其次，骨骼肌中亦有分布，对照组小鼠组织切片未检测到荧光。
第二部分pTAT—BDNF载体构建、表达及TAT—BDNF对多巴胺能神经元的营养作用
方法：
1．提取人脑组织RNA，设计合成引物，RT—PCR的方法克隆人BDNF基因。
2．将酶切的BDNF基因与原核表达载体pTAT／HA相连接，构建重组表达载体pTAT／HA—BDNF。双酶切及基因测序鉴定重组质粒。
3．将质粒转至大肠杆菌E．coli BL21(DE3)中，在IPTG的诱导下，优化表达条件，表达出融合蛋白TAT—BDNF。SDS—PAGE凝胶电泳检测蛋白的分子量。
4．包涵体蛋白经过洗涤、变性溶解后过Ni离子亲和层析柱纯化。
5．Western-blot进一步检测融合蛋白TAT—BDNF的完整性。
6．取14d孕鼠，分离培养胎鼠的中脑神经元细胞，用TH及MAP2免疫荧光细胞化学染色鉴定多巴胺能神经元细胞。
7．原代培养多巴胺能神经元细胞，1w换2次液，观察细胞的生长状况。
8．原代培养至第6d，分成5组，第1、2组暂不加干预因素，第3、4、5组加入不同浓度的TAT—BDNF，至第10d，除第一组外，均加入100μmol／L的6～0HDA，继续培养24h后，通过MTT检测细胞的活力，来观察TAT—BDNF对多巴胺能神经元的保护作用。
9．各组细胞在加入6—OHDA 24h后，Hoechst33258／PI双重荧光染色检测细胞凋亡，观察TAT—BDNF对多巴胺能神经元的保护作用。
结果：
1．常规提取出人脑RNA，用巢式PCR的方法得到约763bp的人BDNE基因，与预期相符。
2．质粒pTAT／HA—BDNF用EcoRⅠ和XhoⅠ酶切鉴定，切出与BDNF的PCR产物大小相当的片段，以及与载体pTAT／HA(3.0kb)大小一致的片段，表明人BDNF基因已正确插入载体pTAT／HA。pTAT／HA—BDNF重组质粒测序结果与Gnebank登录的基因序列相比，序列完全相符，表明成功得到重组载体pTAT／HA—BDNF。
3．重组pTAT／HA—BDNF转化BL21(DE3)菌后，经IPTG诱导后产生一条约35KD的特异蛋白条带，与预期的蛋白质分子量大小相符。诱导条件优化后发现，在IPTG为0.5mmol，37℃，4h的条件下诱导的目标蛋白总蛋白比值最高。
4．蛋白几乎都是以包涵体的形式存在于沉淀中，包涵体经过洗涤，变性溶解后，经过镍离子亲和层析柱纯化得到较为纯净的蛋白。
5．Western—blot检测TAT—BDNF同时具有BDNF的免疫原性及多聚组氨酸的免疫原性，说明表达的融合蛋白完整。
6．TH及MAP2免疫荧光细胞化学染色鉴定多巴胺能神经元细胞，发现多巴胺能神经元细胞占80％以上，完全能达到实验的需要。
7．原代培养的不同天数观察细胞，可见到细胞贴壁，长出神经突起并延长交织呈网状。
8．四唑盐(MTT)比色法测定细胞生长指数，5、20、50μg／L TAT—BDNF预处理组多巴胺能神经元细胞活力较6—OHDA组比均有显著提高(P＜0.01)，而与正常对照组比较无明显差异，其中50、20μg／L TAT—BDNF较5μg／L TAT—BDNF细胞活力更高(P＜0.05)。
9．Hoechst33258／PI双重荧光染色可以看到，正常培养的多巴胺能神经元仅有少量的细胞凋亡，经6—OHDA作用后可见大量的细胞凋亡，经过TAT—BDNF预处理的细胞凋亡的情况较6—OHDA作用组明显减少，而50μg／ml、20μg／ml实验组较5μg／ml实验组效果明显。
第三部分融和蛋白TAT—BDNF对PD大鼠的治疗作用
方法：
1．用6—OHDA与立体定向仪，制作PD大鼠纹状体二点单侧毁损模型。
2．检测TAT—BDNF在脑内的分布：大鼠随即分为2组：实验组及对照组，每组3只，共6只。实验组在制作模型当时，尾静脉注射0.1ml(500μg)的TAT—BDNF，转导对照组尾静脉注射0.1ml的生理盐水，4h后取中脑组织，用HA免疫荧光组织化学染色来检测TAT—BDNF是否进入脑组织内。
3．提取实验组及对照组大鼠中脑组织，Western—blot检测TAT—BDNF在脑组织内的分布。
4．TAT—BDNF对PD大鼠的治疗作用：大鼠随即分为4组：正常组，模型组，实验组及对照组，每组16只，共64只。实验组在制作模型当时，尾静脉注射0.1ml(500μg)的TAT—BDNF，以后每7d注射一次，至28d。对照组尾静脉注射0.1ml的生理盐水，每7d注射一次，至28d。各组在不同的时间点作行为学检测，记录并比较阿扑吗啡诱发的旋转圈数。
5．第28d时，TH免疫组织荧光检测各组大鼠黑质中多巴胺能神经元，计算细胞核轮廓清晰的TH免疫阳性神经元，比较各组之间的差异。
6．第28d时，TUNEL法检测大鼠中脑黑质凋亡神经元，计算每张冠状切片的TUNEL阳性细胞数，比较各组之间的差异。
结果：
1．采用荧光免疫组织化学染色后在荧光显微镜下观察，静脉注射TAT—BDNF蛋白4h后PD大鼠的中脑内能看到均匀分布的由HA标记的融合蛋白，而对照组在荧光显微镜下不能看到荧光标记的TAT—BDNF，证实TAT能介导BDNF穿过血脑屏障进入中枢神经系统。
2．Western—blot分析结果显示在注射TAT—BDNF融合蛋白4h后的大鼠中脑的蛋白提取液中能检测到具有多聚组氨酸抗原特性的蛋白质。而从转导对照组大鼠中脑提取的蛋白质则不能与多聚组氨酸抗体起反应。说明融合蛋白已穿过血脑屏障分布到中枢神经组织内。
3．模型组和对照组大鼠旋转行为最为明显，实验组转速有所下降，旋转的幅度也减轻，表现为旋转的圆周扩大，而正常组无明确的旋转行为。在实验组治疗的前14d统计结果显示实验组和对照组之间没有差异，第21d时两组有显著性差异，第28d时两组比较有极显著差异。
4．正常组大鼠黑质内TH阳性神经元主要分布于黑质网状部，沿黑质区长轴方向排列，数量较多，胞体较大，呈锥体形或椭圆形，纤维细长，量多而密集神经元突起较明显。模型组TH阳性神经元明显减少，分布稀疏，神经元胞体轮廓及突起不清晰。TAT—BDNF实验组TH阳性神经元及阳性纤维较对照组明显改善，神经元及纤维数量明显增多。
5．TUNEL法显示模型组及对照组大鼠损毁侧中脑黑质区有大量染色阳性细胞，细胞核固缩，呈棕黄色，并可见裂解的棕色凋亡小体。通过计算机图像分析系统统计平均每张冠状切片的凋亡细胞数。结果显示损伤后注射TAT—BDNF蛋白的大鼠平均每张中脑黑质切片的TUNEL阳性细胞数明显小于对照组，正常组未见阳性细胞。说明6—OHDA注入纹状体后可逆向引发同侧黑质DA能神经元以凋亡形式发生退化，TAT—BDNF可能通过抗凋亡的作用保护大鼠多巴胺能神经元。
结论
1．小鼠尾静脉注射融合蛋白TAT—EGFP 4h后，在肝、脑、肾、脾、心肌及骨骼肌等组织内均有绿色荧光的分布，而注射了EGFP的对照组组织中未见荧光，提示了TAT能够携带EGFP穿透生物膜屏障进入各个组织，甚至是穿透血脑屏障进入中枢神经系统，为TAT携带大分子蛋白物质治疗中枢神经系统疾病提供了理论基础。
2．首次将表达的融和蛋白TAT—BDNF与多巴胺能神经元作用后，证实TAT—BDNF能保护细胞抵抗6—OHDA的神经毒性，细胞存活率明显提高。提示合成的TAT—BDNF具有与BDNF相同的神经保护的生物学活性。
3．采用纹状体二点单侧毁损术成功制备了PD大鼠模型，从大鼠旋转行为的变化可看出6—OHDA的对黑质内DA能神经元的毁损是渐进性的，比黑质毁损法制备的模型能更好地模拟PD的发病过程。提示该模型能更好地显示在PD早期DA能神经元进行性变性阶段药物的治疗作用。
4．首次将融和蛋白TAT—BDNF用于PD大鼠模型的治疗，实验组大鼠较对照组在行为学上有很大的改善，TH阳性神经元增多，TUNEL染色显示凋亡细胞减少。提示TAT能携带BDNF穿透血脑屏障进入中枢神经系统起到治疗作用，给临床外源大分子物质治疗PD提供了一条新思路。
Parkinson 's disease (PD) is a common chronic degenerative disease of central nervous system (CNS) in the middle—aged and old people. It is also one of the main causes of disability for these people. The pathology of this disease is retrograde degeneration of dopaminergic neuron in the substantia nigra of midbrain and striatum which result in the functional disorder of transmitter system in striatum system. Clinically, cardinal symptoms are static tremor, movement retardation, hypokinesia, rigidity and posture balance disturbance and so on. Incidence and disability of PD are high, which puts a heavy burden on society,family and individual.At present, only symptomatic treatment can not prevent dopaminergic neuron from degenerating. Transplantation of embryonic stem cells is limited because of insufficient materials source and the low survival rate of transplanted cells. Gene therapy is also faced with the problems of safety and toxicity. So it is very important to find some new agents and medicines to prevent and treat PD.
Brain derived neurotrophic factor(BDNF) belongs to the family of neurotrophin. It can promote many kinds of neurons to survive, especially the strong function of nutrition and protection to the dopaminergic neuron. BDNF can not only promote survival differentiation and growth of the dopaminergic neuron but also prevent dopaminergic neuron from degeneration caused by neurotoxicity substance.So BDNF could be a new kind of drug to treat Parkinson. But BDNF is macromolecular substance which can not permeate blood brain barrier(BBB), so it is difficult to apply clinically. TAT is trans—activator of HIV—1,and it can transmembrane into cell and transduct the covalent—bonding heterogenic protein into cell, At the same time, it also can permeate the BBB. Foreign scholars had fused TAT with protein whose molecular weight was (15～200)×10~3 and found these fusion proteins could been transducted into cells. It was reported that TAT fused SOD—Cu,Zn could transmembrane the beta Cell of the islet to treat diabetes. Also TAT fused the p53 and p27 can lengthen the life span of the mice which suffered from the tumor. At present, the study of TAT fusion protein in the field of nerves system focuses on cerebral ischemia. There is no report that the TAT fusion protein treats the PD.
We firstly utilized the enhanced green fluorescent protein(EGFP) as marker, constructed the plasmid TAT—EGFP and expressed the fusion protein, observing the protein distribution in different tissues after intravenous injecting the protein into mice, so that we can confirm the protein transduction ability, especially the ability of permeating BBB. Facts mentions aboveprovided the theory foundation of TAT mediates medicine to treat central nervous system disease. Then we constructed the plasmid TAT—BDNF and expressed the fusion protein, after detecting its neuroprotective activity by incubation with dopaminergic neuron in vitro. TAT—BDNF was intravenously injected into rat model of PD. Its neuroprotective effect was evaluated by observing the ethology and histomorphology of rats.in order to find a convenient and effective treatment for PD.
The Study Includes Three Parts:
PartⅠTAT Mediates EGFP to Transmembrane Transport into Mice Tissues
Methods
1. With the template pEGFP-N3, pEGFP was constructed by PCR and gene cloning technique.
2. Double strands was obtained by annealing the two synthesized DNA fragment encoding TAT protein transduction domain, then it was ligated to pEGFP after digestion by restriction enzyme to construct the expression vector of pTAT—EGFP.
3. pTAT—EGFP was transformed into E.coli BL21 (DE3), the fusion protein was expressed by IPTG. Molecular weight was detected by SDS—PAGE gel electrophoresis. Protein EGFP was obtained also in this step.
4. The protein was purified by Ni~(2+) affinity column.
5. The fusion protein was intravenously injected into mice by vena caudalis. Four hours later, frozen section was made from brain, heart muscle, liver, spleen, kidney and so on. Distribution of the fusion protein in different tissues was observed in fluorescence microscope.At the mean time, EGFP protein was intravenously injected into the control group mice.
Results
1. 720bp PCR product was confirmed by agarose gel electrophoresis, which was in line with the expectancy of EGFP.
2. The enzyme digestion products of recombinant plasmid pEGFPand pTAT—EGFP are 720bp and 5000bp, it is consistent with the expectancy of EGFP and pET28a. After gene sequencing, the recombinant plasmid sequence is 100% consistent with the sequence in Genebank. It showed that the EGFP gene was inserted the plasmid correctly, the recombinant plasmid were obtained successfully.
3. induced by IPTG, the expressed fusion protein TAT—EGFP was maximum in 0.5 mmol/L IPTG、30℃and 3h, in the same way, the expressed protein EGFP was maximum in 0.5 mmol/L IPTG, 30℃and 4h. Molecular weight of TAT—EGFP and EGFP are 28KD and 27KD by SDS-PAGE gel electrophoresis detection.
4. The purity coefficient of protein exceeds 85% after purification by Ni~(2+) affinity column.
5. Four hours later, in experimental group mice, green fluorescence was detected in different tissues, including brain, heart muscle, liver, spleen, kidney and so on. Among them, the fluorescence in liver and kidney were the most obvious, while in heart muscle and brain were moderate, and so did in skeletal muscle. In control group mice no fluorescence were detected in all tissues.
PartⅡExperiment Study of Construction and Expression of pTAT—BDNF and the Neuroprotective Effect of TAT—BDNF to Aminergic Neuron
Methods
1. RNA was extracted from human brain tissue, and human BDNF gene was clonedby RT—PCT.
2. BDNF gene was ligated to prokaryotic expression vector pTAT/HA afterdigestion by restriction enzyme,then the recombinant expression vector of pTAT/HA—BDNF was constructed.The recombinant vector was identified by double restriction enzyme digestion and gene sequencing.
3. pTAT/HA-BDNF was transformed into E.coli BL21 (DE3), the fusion protein TAT—BDNF was expressed by IPTG. Molecular weight was detected by SDS—PAGE gel electrophoresis.
4. Inclusion was purified by Ni~(2+) affinity column after washing and denaturation.
5. Inclusion body of the fusion protein TAT—BDNF was further detected by Western—blot.
6. Embryos were taken out from the pregnant mice, the midbrain neurons were isolated and cultivated. Dopaminergic neuron was identificated by TH and MAP2 double immunofluorescence stain.
7. Dopaminergic neurons were primary cultivated, and nutrient medium was exchanged twice a week. The growth state was observed by microscope.
8. Cells were divided into 5 groups when cultivated in the sixth day, and different concentration TAT—BDNF was added into the third, the forth and the fifth group. In the tenth day, except for the first group, 100μg 6—OHDA was added into all groups, then cells continue to been cultivated for 24 hours. Protection to dopaminergic neuron was observed by detecting cell vigor using MTT method.
9. Apoptosis was detected by hoechst 33258 and PI double immunofluorescence stain after 6—OHDA was added.
Results
1. Human BDNF gene of 763bp was obtained by nest PCR from the human brain,it was in line with the expectancy of BDNF.
2. The product of pTAT/HA—BDNF digestedby restriction enzyme was about 720bp and 3.0kb which is in line with the BDNF gene and the pTAT/HA vector, so it showed that human BDNF had already inserted the vector correctly. Gene sequencing confirmed that pTAT/HA—BDNF according with the sequence in Gene bank. The recombinant vector was obtained successfully.
3. pTAT/HA—BDNF was transformed into E.coli BL21 (DE3), and SDS—PAGE gel electrophoresis showed the protein of 35KD was produced by IPTG By changing the expression condition, maximum fusion protein was expressed.
4. Most of expressed protein was reside in the sediment in the style of occlusion body, occlusion body was purified by Ni~(2+) affinity column after washing and denaturation.
5. TAT—BDNF had the immunogenicity of both poly—histidine and BDNF by Western—blot, which showed that the fusion protein was integrity.
6. It was identified that dopaminergic neuron were above 80% in the midbrain neurons by TH and MAP2 double immunofluorescence stain. This satisfied us that it is enough for the next step.
7. The dopaminergic neuron was observed in different time, cell adherence,nervous process eruption and extension into reticulation can be observed by microscope.
8. Cell growth index was detected by MTT chromatometry, the result showed that cell survival rate in the group of pre—disposal treatment using different concentration TAT—BDNF increased significantly than that of 6—OHDA group.Futhermore, the cell survival rate of 50, 20μg BDNF is higher that of 5μg BDNF.
9.Slight apoptosis can be observed in normally cultivated dopaminergic neurons by Hoechst 33258 and PI double immunofluorescence stain,a lot of cell apoptosis in 6—OHDA group, apoptosis in the group of pre—disposal TAT—BDNF treatment decreased obviously than that of 6—OHDA group. Furthermore, neuroprotective effect of 50μg/ml and 20μg/ml is more significant than that of 5μg/ml treatment.
PartⅢThe Therapeutical Effect of Fusion TAT—BDNF on Rat Model of PD
Methods
1 Using stereotaxic apparatus, PD models were made by injecting the 6—OHDA into unilateral Striatum of the rats.
2 Distribution detection of the TAT—BDNF: Rats were randomly divided into two groups—study group and control group, and there are 3 rats in each group. Study group rats were intravenously injected 0.1ml(500μg) TAT—BDNF by vena caudalis when rats were made into the PD models, rats of control group were injected 0.1ml physiological saline. Four hours later, rats were killed and midbrain tissues were taken out, whether or not TAT—BDNF got into brain tissue was observed by HA immunofluorescence histochemical stain.
3 Midbrain tissues of rats were extracted and TAT—BDNF was identified by Western—blot so that we can confirm that TAT—BDNF distributed into brain tissues.
4 Neuroprotective effect of TAT—BDNF rats model of PD: Rats were randomly divided into four groups—normal group,model group, study group, control group. There were 16 rats in each group. Study group rats were intravenously injected 0.1ml(500μg) TAT—BDNF by vena caudalis when they were made into the PD models, then every 7day to 28 day, same dosage TAT—BDNF was injected into Study group rats. Control group rats were injected 0.1ml physiological saline. In different time, rats of every group were examined in ethology, and rotation circle numbers were record and compared.
5 Dopaminergic neurons that TH immunization was positive were counted by TH immunohistofluorescence in 28th day. Number of dopaminergic neurons in different group was compared.
6 Substantia nigra of midbrain was detected by TUNEL, and TUNNEI positive cells were counted and compared in the different groups.
Results
1 Using the HA immunofluorescence histochemical stain method, we saw the fusion protein homogeneous distribution in mid—brain of rat by fluorescence microscope, However, no fluorescence was detected in the rats of control group, which confirmed that TAT can mediate BDNF to permeate the BBB in CNS of rats.
2 Western-blot analysis showed that there was fusion protein in the midbrain of rats which was injected the TAT—BDNF,however, there was no fusion protein in the midbrain of control rats. It indicated that fusion protein had already permeated the BBB into central nervous system of rats.
3 The rotational behavior of study group rats was distinctly different from model group threats. The rotation speed of study group rats was decrease,and the extent of rotation was lighten. Rats of normal group did not rotate. Statistical results showed that the rotational behavior in four group rats was not discrepancy in first two weeks. However, in the third week, the rotational behavior of the study group rats was significantly different from the control group rats,and in the forth week, the difference was extreme in two group rats.
4 In normal rats, there were many large cells of TH—immunoreactive(IR) positive located mainly in the reticular compacta of substantia nigra, arranging along the long axis of substantia nigra region. These cells were pyramid or ellipse and their fibers were slender. TH positive cells were very few in rats of model group, cells distributed sparseness and their outline and neurite were fuzziness. TH positive cells and fibers were increased significantly in rats of study group, and the outline and neurite of neuros were distinct.
5 A great quantity of positive cells in substantia nigra of midbrain were observed in the rats of model group and the control group by TUNEL, these cell nucleuses were buffy and pyknosis, and brown apoptotic bodies were lytic. Analysis results showed that the positive cells in study group rats were less than that of control group significantly. There were not positive cells in normal group rats. It illustrated that the degeneration of DA neurons was related with apoptosis after 6—OHDA was injected into striatum of rats, and TAT—BDNF can keep DA neurons from apoptosis.
Conclusions:
1 Green fluorescence was detected in different tissues in study rats,such as brain, heart muscle, liver, spleen, kidney and so on, After injecting TAT—EGFP by vena caudalis while no fluorescence was detected in all tissues of control group mice, which indicated that TAT can mediated EGFP to permeat into tissues of mice, especially into CNS. These results provide us a new way to treat CNS disease using large molecular substance.
2 Fusion protein TAT—BDNF that we expressed could protect the dopaminergic neurons from the neurotoxicity of 6—OHDA, which indicated that TAT—BDNF has the neuroprotective activity.
3 We made the rats model of PD by two point injecting 6—OHDA into unilateral striatum of the rats successfully. The damage of 6—OHDA to dopaminergic neurons is gradually, so this model was more similar to the process of Parkinson disease than the models with other methods. Which indicated that this model is available for observing the therapeutic effect of medicine at the stage of progressive degeneration of neurons.
4 When fusion protein TAT—BDNF was used to treat the rats model of PD, we found that the ethology of study group rats was improved more obviously than the control group rats. There were more TH positive neurons and less TUNEL positive neurons in study group rats than the control group rats. These illustrated that TAT—BDNF could permeat BBB into the central nervous system, and TAT—BDNF had the neuroprotetive effect to dopaminergic neuron.
引文
1 徐德隆，陈生弟，刘振国，主编．帕金森病临床新技术．北京，人民军医出版社，2002年，第1版，49～50．
2 Simpkins N,Jankovic J.Neuroprotection in Parkinson Disease[J].Arch Intern Med,2003,163 (14):1650～1654.
3 Shumsky JS,Tobias CA,Tumolo M,et al.Delayed transplantation of fibroblasts genetically modified to secrete BDNF and NT—3 into a spinal cord injury site is associated with limited recovery of function[J].Experimental Neurology, 2003,184(1):114～130.
4 Von Bartheld CS,Johnson JE. Target- derived BDNF (brain- derived neurotrophic factor) is essential for the survival of developing neurons in the isthmo—optic nucleus[J].J Comp Neurol,2001,433(4):550～564.
5 Green M,Loewenstein PM.Autonomous functional domains of chemically synthesized human immunodeficiency virus Tat trans—activator protein[J].Cell, 1988,55(5): 1179～1188.
6 Frankel AD,Pabo Co. Cellular uptake of the Tat protein from human immuno deficiency virus[J].Cell,1988,55 (5) :1189～1193.
7 Sik Eum M,Soon Choung I,Zhen Li M,et al.HIV—1 tat mediated protein transduction of Cu,Z—superoxide dismutase into pancreaticβcells in vitro and in vivo[J].Free Radical Biology &Medicine,2004,37(3):339～349.
8 Becker—HapakM, McAllister SS, Dowdy SF. TAT mediated protein transduction into mammalian cells[J].Methods,2001,24 (3) :247～256.
9 Okada CY,Rechsteiner M.Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles[J].Cell,1982,29(1):33-41.
10 Chakrabarti R,Wylie DE,Schuster SM.Transfer of monoclonal antibodies into mammalian cells by electroporation[J].J Biol Chem, 1989,264(26): 15494 - 500.
11 Fawell S,Seery J,Daikh Y,et al.Tat-mediated delivery of heterologous proteins into cells[J].Proc. Natl Acad Sci USA,1994,91(2):664-668.
12 Vives E,Brodin P,Lebieu B.A truncated HIV- 1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus[J].J Biol Chem, 1997,272(25): 16010 - 16017.
13 Steven R S,ALan Ho,Adamina V et al .In vivo protein transduction : delivery of a biologically active protein into the mouse[J].Science, 1999,285(5433): 1569- 1572.
14 Futaki S,Suzuki T,Ohashi et al.Arginine-rich peptides.An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery[J].J Biol Chem,2001;276(8):5836-5840.
15 Brooks H,Lebleu B,Vives E.Tat peptide-mediated cellular delivery: back to basics[J].Adv Drug Deliv Rev, 2005;57(4):559-577.
16 Trehin R,Merkle HP.Chances and pitfalls of cell penetrating peptides for cellular drug delivery[J]. Eur J Pharm Biopharm,2004,58(2):209-223.
17 Rothbard JB,Jessop TC,Lewis RS, et al. Role of membrane potential and hydrogen bonding in the mechanism of translocation of guanidinium - rich peptides into cells[J].J Am Chem Soc,2004,126(31):9506-9507.
18 Schwarze.SR,Ho A,Vocero - Akbani A,et al.In Vivo Protein Transduction: Delivery of a Biologically Active Protein into the Mouse science, 1999, 285:1569-1572.
19 Jin LH,Bahn JH,Eum WS et al .Transduction of human catalase mediated by an HIV- 1 TAT protein basic domain and arginine-rich peptides into mammalian cells. Free Radic Biol Med,2001,31(11): 1509-1519.
1 Shumsky JS,Tobias CA,Tumolo M,et al.Delayed transplantation of fibroblasts genetically modified to secrete BDNF and NT—3 into a spinal cord injury site isassociated with limited recovery of function[J].Experimental Neurology,2003, 184(1):114～130.
2 Von Barthelld CS,Johnson JE.Target—derived BDNF (brain—derived neurotrophic factor) is essential for the survival of developing neurons in the isthmo—optic nucleus[J].JComp Neurol,2001,433(4):550～564.
3 Shults CW,Kinker T.BDNF attenuates the effects of intranstraital injection of 6 —hydroxydopamine[J].Neurorepor, 199,6(8): 1109.
4 Iskandrian AS. Adenosine myocardial perfusion imaging[J]. J Nuel Med ,1994, 35(4): 734.
5 Minichiello L,Calella A,Medina D,et al.Mechanism of TrkB—mediated hippocampal long—term potentiation[J].Neuron,2002,36(1): 121.
6 Green M,Loewenstein PM.Autonomous functional domains of chemically synthesized human immunodeficiency virus Tat trans—activator protein[J]. Cell,1988, 55(5):1179～1188.
7 Frankel AD,Pabo Co.Cellular uptake of the Tat protein from human immunodeficiency virus[J].Cell, 1988,55(5): 1189～1193.
8 Vives E,Brodin P,Lebieu B. A truncated HIV—1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus[J].J Biol Chem, 1997,272(25): 16010～16017.
9 Schwarze SR,Ho A,Vocero--Akbani A,et al.In Vivo Protein Transduction: Delivery of a Biologically Active Protein into the Mouse[J].science,1999, 285(5433): 1569～1572.
10 Becker—HapakM, McALLister SS,Dowdy SF. TAT mediated protein transduction into mammalian cells[J].Methods,2001,24 (3) :247～256.
11 Barde Y A, Edgar D, Thoenen H, Purification of new neurotrophicfactor from brain [J] EMBO J,1982,1(5):549v553.
12 Ivanova T, Beyer C, Preo and postnatal expression of brain derived neurotro— phic foctor mRNA/protein and tyrosine protein kinage receptor B mRNA in the mouse hippocampos[J]. Neurosci lett,2001,307(1):21—24.
13 李力燕，王廷华等．脑源性神经营养因子在成年猴脑的分布．神经解剖学杂志，2002(1)：67—70．
14 Zhou J,Bradford HF, Stern GM,et al.The response of human and rat fetal ventral mesencephalon in culture to the brain—derived neurotrophic factor treatment[J]. Brain Res, 1994,656(1): 147～156.
15 Studer L, Spenger C, Seiler RW, et al. Effects of brain—derived neurotrophic factor on neuronal structure of dopaminergic neurons in dissociated cultures of human fetal mesencephalon[J].Exp Brain Res, 1996,108(2):328～336.
16 Michel PP, Ruberg M, Agid Y. Rescue of mesencephalic dopamine neurons by anticancer drug cytosine arabinoside[J].J Neurochem, 1997,69(4): 1499～1507.
17 Hoglinger GU, Sautter J, Meyer M, et al. Rat fetal ventral mesencephalon grown as solid tissue cultures: influence of culture time and BDNF treatment on dopamine neuron survival and function[J].Brain Res, 1998,813:313～322.
18 Spenger C, Hyman C, Studer L,et al. Effects of BDNF on dopaminergic, serotonergic, and GABAergic neurons in cultures of human fetal ventral mesencephalon[J].Exp Neurol, 1995,133(2):50～63.
19 Beck KD,Knusel B,Hefti F.The nature of the trophic action of brain—derived neurotrophic factor, des (1—3)--insulin--like growth factor--1, and basic fibroblast growth factor on mesencephalic dopaminergic neurons developing in culture[J].Neuroscience,1993,52(4):855～866.
20 Kontkanen O, Castren E. Trophic effects of selegiline on cultured dopaminergic neurons[J].Brain Res, 1999,829(1—2): 190～192.
21 Skaper SD,Negro A,Facci L,et al.Brain—derived neurotrophic factor selectiveLy rescues mesencephalic dopaminergic neurons from 2,4,5- trihydroxyp henylalanine—induced injury[J].J Neurosci Res,1993,34(4):478～487.
22 Spina MB, Squinto SP, Miller J,et al. Brain—derived neurotrophie factor protects dopamine neurons against 6—hydroxydopamine and N—methyl—4— phenylpyridinium ion toxicity: involvement of the glutathione system. J Neurochem[J], 1992,59(1):99～106.
23 Menei P , Whittemor S R .Schwann cells genetically modified to secrete human BDNF promote enhanced axonal regrowth across transected adult rat spinal cord[J].European Journal of Neuroscience, 1998,10(2):607～621.
24 Rubio F, Kokaia Z,Arco A, et al.BDNF gene transfer to the mammalian brain using CNS—derived neural precursors[J].Gene Ther, 1999, 6(11): 1851～1866.
25 Maisonpierre PC,Belluscio L,Squinto S,et al.Neurotrophin3:A neurotrophic factor related to NGF and BDNF[J].Science, 1990,247:1446～1451
26 Nagahara H, Snyder E.L, Snyder EL, et al.Transduction of full—length Tat fusion proteins into mammalian cells:TAT—p27Kipl induces cell migration[J].Nature Medicine. 1998,4(12): 1449～1452.
27 Berger B,Diporzio U,Daguet MC,et al.Long term development of mesencephalic dopaminergic and non—dopaminergic neurons in organotypic slice cultures of the rat ventral mesencephalon[J].ExpBrain Res, 1995,106(2):205～214.
28 Jia XJ,Gong SL, Liu SZ,et al.Dopaminergic neuron as the cell model for Parkinson's disease[J].J Chin J Neuromed,2004,3(5):382～386.
29 苏雅茹，蒋雨平．6—多巴胺在帕金森病发病中的作用[J]．中国临床神经科学．2002，10(3)：312～314．
30 Nass R,Hall DH,Miller DM,et al.Neurotoxin—induced degeneration of dopamine neurons in Caenorhabditis elegans[J].Neurobiology,2002,99(5): 3264～3269.
31 马敬红，胡国华，董丽华，等．6—羟基多巴在帕金森病发病机制中的作用[J]．吉林大学学报：医学版，2005，31(3)：355～357．
32 Zhang J, Liu X.Study on tetrazolium salt colorimetric assay for growth and survival of bacteria[J].Wei Sheng Yah Jiu,2002,31(5):361～363.
1 徐德隆，陈生弟，刘振国，主编．帕金森病临床新技术[J]．北京，人民军医出版社，2002年，第1版，p49～50．
2 Simpkins N,Jankovic J.Neuroprotection in Parkinson Disease[J].Arch Intern Med, 2003,163(14):1650～1654.
3 Green M,Loewenstein PM.Autonomous functional domains of chemically Synthesized human immunodeficiency virus Tat trans — activator protein[J].Cell,1988,55(5): 1179～1188.
4 Frankel AD,Pabo CO.Cellular uptake of the Tat protein from human immunodeficiency virus[J].Cell, 1988,55:1189～1193.
5 Fawell S,Seery J,Daikh Y, et al. Tat—mediated delivery of heterologous proteins into celLs[J].Proc. Natl Acad Sci USA, 1994,91 (2):664～8.
6 Perry TL, Ito M. Nigrostriatal dopam inergicneurons remain undamaged in rats given high doses of L — DOPA and caxbidopa chronically[J]. Neurochem, 1984,43(3):990～3.
7 包新民，舒斯云,主编．人鼠脑立体定位图谱[M]．北京人民卫生出版社，1991年，第1版,49～58．
8 9 Daly TM,Okuyama T, Vogler C,et al.Neonatal intramuscular injection with recombinant adeno — associated virus results in prolonged beta—glucuronidase expression in situ and correction of liver pathology in mucopolysac—charidosis type Ⅶ mice[J].Hum.Gene Ther, 1999,10(1):85～94.
9 盛树力，临床神经科学前沿[M]．北京：北京大学医学出版社,2003,20(7)：5411～5417．
10 Hattori N,Machida Y, Noda K. Pathogenesis of Parkinson's disease: a common pathway between alpha—synuclein and parkin and the mechanism of lewy bodies formation[J].Rinsho Shinkeigaku,2005,45(11):905～907.
11 Backlund EO, Granberg PO, Hamberger B, et al. Transplantation of a adrenal medullary tissue to striatum in Parkinsonism first clinical trials[J].Neurosurg, 1985,62(2): 169～173
12 Widner H,Tetrud J,Rehncrona S,et al.Bilateral fetal mesencephalic grating in two patients with parkinsonism induced by 1—methyl—4—phenyl—1,2,3,6—tetr ahydropyridine(MPTP)[J].Nat Eng J Med, 1992,327(22): 1556～1563
13 Lindvall O.Update on fetal transplantation:the Swedish experience [J].Mov.Disord, 1998,13:83～87.
14 Li JY, Brundin P. Grafting dopamine neurons in Parkinson's disease: Do stem cells have a role in the future? [J].Neurochem,2003,85(5): 13～15.
15 Mark S, Meena J,Roger A.The cellular repair of the brain in Parkinson's disease —past,present and future[J].Transplant Immunology,2004,12(3—4):321～342
16 陈生弟主编．帕金森病临床新技术[M]．人民军医出版社．2002年第1版．140～223．
17 Ronald D,Arjan B,Jos P.Modeling Parkinson's disease in rats:An evaluation of 6 —OHDA lesions of the nigrostriatalpathway[J].Experimental Neurology, 2002,175(2):303～317.
18 赵焕英，苏月，杨秋慧等．6—羟基多巴胺纹状体内注射制作大鼠帕金森病模型的研究[J]．中国组织化学与细胞化学杂志，2003，12(1)：16～21
19 Darwin J,Prockop,Carl A,et al.One strategy for cell and gene therapy:Harnessing the power of adult stem cells to repair tissues[J].PNAS September,2003, 100:11917～11923
20 Prockop DJ.Marrow Stromal Cells as Stem Cells for Momhematopoietic tis sues[J].Science, 1997,276:71～74
21 Deari C,Dell'Accio F, Vandenabeele F, et al.Skeletal muscle repair by a—dolt human mesenchymal stem cells from synovial membrane[J].Cell Biol,2003, 160(6):909～9184
22 Sanchez Ramos J,Song S,Cardozo Pelaez F, et al.Adult bone marrow stromal cells diferentiate into neural cells in vitro[J].Exp Neurol,2000,164(2):247～256
23 Woodbury D,Schwarz EJ,Prockop DJ,et al.Adult rat and human bone marrow stromal cells diferentiate into neurons[J].Neurosic Res,2000,61 (4):364～370
24 Azizi SA,Stokes D,Augelli B,et al. Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats similarities to astrocyte grafts[J]. Proc Natl Acad Sci USA,1998,95(7):3908～3913
25 Mezey E,Chandross KJ,Harta G, et al.Turning blood into brain:cells bearing neuronal antigens generated in vivo from bone marrow[J].Science,2000, 290(5497): 1779～1782
27 Meakin SO,Shooter EM.The growth factor family of receptors[J].Trends Neurosci, 1992,15:323～331
27 Orlic D,Kajstura J,Chimenti S,et al.Bone marrow cells regenerate infarcted myocardium[J].Nature,2001.410(6829):701～705
28 Murphy JM,KavaLkovitch KW, Fink D, et al.Regeneration of meniseal tissue and protection of articult cartilge by onjection of mesenchymalstem cells[J]. Osteoarthritis Cartilage,2000,8:25～29
29 Chen J,Li Y, Wang L,et al. Therapeutic benefit of intravenous administration of bone stromal cells after cerebral ischemia in rata[J].Stroke,2001,32(4):1005～1011
1 Varga CM,Wickham TJ,Lauffenburger DA.Receptor—mediated targeting of gene delivery vectors:insights from molecular mechanisms for improved vehicle design[J].Biotechnol,2000,70(6):593～605.
2 Chakrabarti R, Wylie DE,. Schuster SM.Targeting of gene delivery vectors:insights from molecular mechanisms for improved vehicle design[J].J. Biol. Chem, 1989,264:15494～15500.
3 Arnheiter H, Haller O.Antiviral state against influenza virus neutralized by microinjection of antibodies to interferon—induced Mx proteins[J].EMBO J, 1988,7(5): 1315～1320.
4 Green M,Loewenstein PM.Autonomous functional domains of chemically synthesized human immunodeficiencyvirus TAT trans—activator protein[J].Cell,1988,55 (6) : 1179～1188.
5 Frankel AD,Pabo CO.Cellular uptake of the TAT protein from human immuno deficiency virus[J].Cell,1988,55 (6) :1189～1193.
6 Joliot A, Pernelle C, Deagostini H, et al.Antennapedia homeo box peptide regulates neural morphogenesis[J].Proc Natl Acad Sci, 1991,88(5): 1864～1868.
7 Elliott G., O'Har P.e. Intercellular trafficking and protein delivery by a herpesvirus structural protein[J].Cell. 1997, 88(2):223～233.
8 Lindgren M, Hallbrink M,Prochiantz A, et al.Cell — penetrating peptides[J].Trends Pharmacol.Sci,2000,21 (3):99～103.
9 Hallbrink M,FLoren A,Elmquist A,et al.Cargo delivery kinetics of cell— penetrating peptides[J].Biochim Biophys Acta,2001,1515(2): 101～109.
10 Frankel AD,Bredt DS,Pabo CO.TAT protein from human immuno—deficiency virus forms a metal—linked dimer[J].Science, 1988,240:70～73.
11 Frankel AD, Chen L, Cotter R J, et al. Dimerization of the TAT protein from human immunodeficiency virus:a cysteine—rich peptide mimics the normal metallinkeddimer interface[J].Proc Natl Acad Sci, 1988,85(17):6297～6300.
12 Ruben S, Perkins A, Purcell R, et al.Structural and functional characterization of human immuno deficiency virus TAT protein[J].J Virol, 1989,63(1): 1～8.
13 Siomi H, Shida H, Maki M, et al. Effects of a highly basic region of human immunodeficiency virus TAT protein on nucleolar localization[J] .J Virol, 1990,64(4): 1803-1807.
14 Frankel AD, Biancalana S, Hudson D, Activity of synthetic peptides from the TAT protein of human immunodeficiency virus type l[J].Proc Natl Acad Sci,1989,86(19):397-401.
15 Mann DA, Frankel AD.Endocytosis and targeting of exogenous HIV-1 TAT Protein[J].EMBO J,1991,10(7):1733-1739.
16 Loret E.P,Vives E,Ho PS, et al.Activating region of HIV-1 TAT protein vacuum UV circular dichroism and energy minimization[J].Bioch emistry,1991,30(24):6013-6023.
17 Ruben S,Perkins A,Purcel Rl,et al.Structural and functional characterization of human immuno deficiency virus TAT protein[J].J Virol, 1989,63(1):1 -8.
18 Schwarze SR, Hruska KA, Dowd SF.Protein transduction unrestricted delivery into all cells?[J].Trends Cell Biol, 2000,10(7):290-295.
19 Gehring WJ,Affolter M, Burglin T. Homeodomain proteins[J].Annu Rev Biochem, 1994,63:487-526.
20 Derossi D, Jolio AH, Chassaing G, et al. The third helix of the Antennapedia homeodomain translocates through biological membranes[J].J Biol Chem, 1994,269(14): 10444- 10450.
21 Elliott G, O'Hare P. Herpes simplex virus type 1 tegument protein VP22 induces the stabilization and hyperacetylation of microtubules[J].J Virol, 1998, 72(8):6448-6455.
22 Aints A,Guven H,Gahrton G,et al.Mapping of herpes simplex virus-1 VP22 functional domains for inter - and sub cellular protein targeting[J].Gene Ther,2001,8(14):1051-1056.
23 Pooga M,Hallbrink M,Zorko M,et al.Cell penetration by transportan[J]. FASEBJ,1998,12(1):67-77.
24 Oehlk JE,Scheller A, Wiesner B,et al.Cellular uptake of an alpha -helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically[J].Biochim Biophys Acta,1998,1414(1 - 2):127-139.
25 Lin YZ, Yao SY. Inhibition of nuclear translocation of trans cription factor NFkappaB by a synthetic peptide containing a cell memb rane permeable motif and nuclear loca - lization sequence[J] . J Biol Chem, 1995,270(24): 14255 - 14258.
26 Oess S, Hildt E.Novel cell permeable motif derived from the PreS2-domain of hepatitis-B virus surface antigens[J].Gene Ther,2000,7(9):750-758.
27 Mi Z,Mai J,Lu X,et al.Characterization of a class of cationic peptides able to faciliTATe efficientprotein transduction in vitro and in vivo[J].Molec Ther, 2000,2(4):339-347.
28 Gorodetsky R,Levdansky L, Vexler A,et al.Liposome transduction into cells enhanced by haptotactic peptides Haptides homologous to fibrinogen C - termini[J]. J Control Release,2004,95(3):477-488.
29 Derossi D, Joliot AH, Chassaing G, et al.The third helix of the antenna pedia homeodomain translocates through biological membranes[J].J Biol Chem, 1994,269:10444-10450.
30 Derossi D,Calvet S,Trembleau A,et al.Cell internalization of the third helix of the antennapedia homeodomain is receptor - independent[J].J Biol Chem, 1996,271(30):18188-18193.
31 Elliott G,O'Hare P,Intercellular trafficking and protein delivery by a herpesvirus structural protein[J].Cell,1997,88(2):223-233.
32 Vives E,Brodin P,Lebleu B.A truncated HIV - 1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus[J].J Biol Chem,1997,272(25):16010-16017.
33 Vogel BE, Lee SJ, HiLdebrand A.A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin[J].J Cell Biol,1993,121(2):461-468.
34 Fender PA.The design, synthesis,and evaluation of molecule:that enable or enhance cellular uptake :peptoid molecular transporters[J].Proc Natl Acad,2000,97(24):l 3003-13008.
35 Vives E,Grannier C,Prevot P,et al.Structure activity relationship study of the plasma membrane translacating potential of a short peptide from HIV-1 Tat protein[J].Lett Peptide, 1997,4:429～436.
36 Futaki S,Suzuki T, Ohashi W, et al.Arginine—rich Peptides:An abundant source of membrane—permeable peptides having potential as carriers for intracellular protein delivery[J].J Biol Chem,2001,276(8):5836～5840.
37 Suzuki T, Futaki S, Niva M, et al.Possible existence of common internalization mechanism among arginine—rich peptide[J].J Biol Chem,2002,277(4):2437～3443.
38 Deros D,Joliot AH, Chassaing G, et al.The third helix of the Antennapedia homeodomain translocate:through biological membrane[J].J Biol Chem, 1994,269:10444～10450
39 Silhol M, Tyagi M, Giacca M, et al.Different mechanisms for cellular internalization of the HIV—1 Tat—derived cell penetrating peptide and recombinant proteins fused to Tat[J].Eur J Biochem,2002,269(2):494～501
40 Schwarze SR, Hruska KA,Dowdy SF.Protein transduction: unrestricted delivery into all cells?[J].Trends Cell Biol,2000,10(7):290～295.
41 Tyagi M, Rusnati M, Presta M, et al.Cell surface heparan sulfate nternalization of HIV—1 tat requiresproteoglycan[J].J Biol Chem,2001,276:3254～3261.
42 Belting M.Heparan sulfate proteoglycan as a plasma membrane carrier[J].Trends Biochem Sci,2003,28(3): 145～151.
43 Silhol M, Tyag M, Giacca M, et al. Different mechanisms for cellular internalization of the HIV—1 Tat—derived cell penetrating peptide andrecombinant proteins fused to Tat[J].Eur J Biochem,2002,269:494～501.
44 Gehring W J, Muller M,Affolter M,et al The structure of the homeodomain and its functional implications[J].Trends Genet, 1990,6(10):323～329.
45 Prochiantz A.Getting hydrophilic compounds into cells:Lessons from homeopeptides [J].Opin. Neurobiol,1996,6(5):629～634.
46 Prochiantz A.Homeodomain—derived peptides:In and out of the cells[J].Ann N Y Acad Sci,1999,886:172～179.
47 Le Roux I, Duharcourt S, Volovitch M, et al. Promoter—specific regulation of gene expression by an exogenouslyadded homedomain that promotes neurite growth[J]. Fed Eur Biochem Soc Lett,1995,368(2):311～314.
48 Drin G, Mazel M, CLair P, et al. Temsamani, Physico-chemical requirements for cellular uptake of pAntp peptide.Role of Lipid - binding affinity[J].Eur J.Biochem,2001,268(5): 1304- 1314.
49 Joliot AH,Triller A,Volovitch M,et al.Lpha-2,8-Polysialic acid is the neuronal surface receptor of antennapedia homeobox peptide[J].New Biol. 1991, 3(11):1121 - 1134.
50 Perez F,Joliot A,Bloch E,et al.Antennapedia homeobox as a signal for the cellularinternalization and nuclear addressing of a small exogenous peptide[J].J Cell Sci,1992,102(Pt4):717-722.
51 Derossi D,Chassaing G, Prochiantz A,Trojan peptides: the penetratin system for intracellular delivery[J].Trends Cell Biol,1998,8:84-87.
52 Berlose JP,Convert O,Derossi D,et al.Chassaing,Conformational and associative behaviours of the third helix of antennapedia homeodomain in membrane- mimetic environments[J].Eur J Biochem, 1996,242(2):372-386.
53 Thoren PE,Persson D,Karlsson M,et al.The antennapedia peptide penetratin translocates across lipid bilayers-the first directobservation[J].Fed Eur Biochem Soc Lett,2000,482(3):265-268.
54 Drin G,Cottin S,Blanc E,et al.Temsamani, Studies on the internalisation mechanism of cationic cell - penetrating peptides[J].J Biol Chem,2003,278: 31192-31201.
55 Elliott GD,Meredith DM.The herpes simplex virus type 1 tegument protein VP22 is encoded by gene[J].J Gen Virol, 1992,73(Pt3):723-726.
56 Kuchler K.Unusual routes of protein secretion: the easy way out[J].Trends Cell Biol,1993,3(12):421-426.
57 Mann DA,Frankel AD.Endocytosis and targeting of exogenous HIV - 1 Tat protein[J].Eur Mol Biol Org, 1991,10(7): 1733 -1739.
58 Frankel AD,Pabo CO.Cellular uptake of the tat protein from human immunodeficiency virus[J].Cell, 1988,55:1189-1193.
59 Rusnati M,Tulipano G,Urbinati C,et al.The basic domain in HIV-1 Tat protein as a target for polysulfonated heparin - mimicking extracellular Tat antagonists[J].J Biol Chem, 1998,273(26): 16027-16037.
60 Brake DA,Debouck C, Biesecker G.Identification of an Arg—GLy—Asp (RGD) cell adhesion site in human immunodeficiency virus typel transacfivation protein tat[J].J Cell Biol, 1990,111 (3): 1275～1281.
61 Ferrari A,Pellegrini V,Arcangeli C,et al.Caveolae—mediated interna lization of extracellular HIV—1tat fusion proteins visualized in real time[J].Mol Ther, 2003,8(2):284～294.
62 Richard JP,Melikov K,Vives E,et al.Cell—penetrating peptides:are—evaluation of the mechanism of cellular uptake[J].J Biol Chem,2003,278(1):585～590.
63 Koppelhus U,Awasthi SK,Zachar V,et al.Cell—dependent differential cellular uptake of PNA,peptides, and PNA—peptide conjugates[J].Antisense Nucleic Acid Drug,2002,12(2):51～63.
64 Lundberg M, Wikstrom S, Johansson M.Cell surface adherence and endocytosis of protein transduction domains[J].Mol Ther,2003,8(1): 143～150.
65 Console S,Marty C,Garcia C—Echeverria, et al.Antennapedia and HIV TAT protein transduction domains promote endocytosis of high Mr cargo upon binding to cell surface gLycosaminoglycans[J].J Biol Chem,2003,278(37): 35109～35114.
66 Sandgren S,Cheng F,Belting M.Nuclear targeting of macro molecular polyanions by an HIV—Tat derived peptide. Role for cellsurface proteoglycans[J].J Biol Chem,2002,277(41):38877～38883.
67 Hallbrink M,Floren A,Elmquist A,et al.Cargo delivery kinetics of cell— penetrating peptides[J].Biochim Biophys Acta,2001,1515(2): 101～109.
68 Rousselle C, Smirnova M, Clair P,et al.Enhanced delivery of doxorubicin into the brain via a peptide—vector—mediatedstrategy: saturation kinetics and specificity[J].J Pharmacol Exp Ther,2001,296(1): 124～131.
69 Fawell S,Seery J,Daikh Y,et al.TAT—mediated delivery of heterologous proteins into cells[J].Proc Natl Acad Sci, 1994,91(2):664～668.
70 Moy P,Daikh Y,Pepinsky B,et al.TAT—mediated protein delivery can facilitate MHC class I presentation of antigens[J].Mol Biotechnol, 1996,6(2): 105～13.
71 Kim DT,Mitchell DJ,Brockstedt DG,et al.Introduction of soluble proteins into the MHC class I pathway by conjugation to an HIV TAT peptide[J].J Immunol, 1997,159(4): 1666～1668.
72 Schwarze SR,Ho A,Vocero—Akbani A, et al.In vivo protein transduction:delivery of a biologically active protein into the mouse[J].Science,1999,285(5433): 1569～1572.
73 Caron NJ,Torrente Y,Camirand G,et al.Intracellular delivery of a TAT-eGFP fusion protein into musclecells[J].Molec Ther,2001,3(3):310～318.
74 Vocero— Akbani A,Lissy N,Dowdy SF.Transduction of full—length TAT fusion proteins directly into mammalian cells:analysis of T cell receptor activation—induced cell death[J].Methods Enzymol,2000,322:508～521.
75 Becker—Hapak M,Mcallister SS,Dowdy SF.TAT mediated protein trans duction into mammalian cells[J].Methods,2001,24(3):247～256.
76 Nagahara H,Vocero—Akbani AM,Snyder EL,et al.Transduction of full—Length TAT fusion proteins intomammalian cells:TAT—p27Kip1 induces cell migration[J].Nat Med,1998,4(12):1449～1452.
77 Embury J,Klein D,Pileggi A,et al.Proteins linked to a protein transduction domain efficiently transduce pancreatic islets[J].Diabetes,2001,50(8): 1706～ 1713.
78 Dolgilevich S,Zaidi N,Song J,et al.Transduction of TAT fusion proteins into osteoclasts and osteoblasts[J].Biochem Biophys Res Commun,2002,299(3): 505～509.
79 Kwon HY,Eum WS,Jang HW,et al.Transduction of Cu,Zn—superoxide dismutase mediated by an HIV—1 TAT protein basic domain into mammalian cells[J].FEBS Lett,2000,485(2—3): 163～167.
80 Jin LH,Bahn JH,Eum WS,et al.Transduction of human catalase mediated by an HIV—1 TAT protein basic domain and arginine—rich peptides into mamma lian cells[J].FreeRadicBiol Med,2001,31(11):1509～1519.
81 Yang Y,Ma J,Song Z,et al.HIV—1 TAT—mediated protein trans duction and subcellular Localization using novel expression vectors[J].FEBS Lett,2002,532 (1-2):36～44.
82 Wheeler DS,Dunsmore KE,Wong HR.Intracellular delivery of HSP70 using HIV —1 TAT protein transduction domain.Biochem[J] .Biophys Res Commun, 2003,301(1):54-59.
83 Yoon HY,Lee SH,Cho SW,et al.TAT-mediated delivery of human glutamate dehydrogenase into PC 12 cells[J].Neurochem,2002,41(1):37-42.
84 Shibagaki N,Udey MC.Dendritic cells transduced with protein antigens induce cytotoxic lymphocytes and elicit antitumor immunity[J].J Immunol, 2002,168(5):2393-2401.
85 Wang HY,Fu T,Wang G,et al.Induction of CD4(+)T cell-dependent antitumor immunity by TAT-mediated tumor antigen delivery into dendritic cells[J].J Clin Invest,2002,109(11):1463- 1470.
86 Kabouridis PS,Hasan M,Newson J,et al.Inhibition of NF-kappa B activity by a membrane transducing mutant of I kappa B alpha[J].J Immunol,2002,169 (5):587-2593.
87 Gustafsson AB,Sayen MR,Williams SD,et al.TAT protein transduction into isolated perfused hearts: TAT— apoptosis repressor with caspase recruit ment domain is cardio protective[J].Circulation,2002,106(6):735-739.
88 Cao G,Pei W,Ge H,et al.In vivo delivery of a Bcl—x1 fusion protein contain ing the TAT protein transduction domain protects against ischemic brain injury and neuronal apoptosis[J].J Neurosci,2002,22(13):5423-5431.
89 Asoh S,Ohsawa I,Mori T,et al.Protection against ischemic brain injury by protein therapeutics[J].Proc Natl Acad Sci,2002,99(26):17107- 17112.
90 Roeder GE,Parish JL,Stem PL,et al.Herpessimplex virus VP22-human papillomavirus E2 fusion proteins produced in mammalian or bacterial cells enter mammalian cells and induce apoptotic cell death[J].Biotechnol Appl Biochem,2004,40(Pt2): 157-165.
91 Derer W,Easwaran HP,Leonhardt H,et al.A novel approach to induce cell cycle reentry in terminally differentiated muscle cells[J].FASEB J,2002,16(1): 132- 133.
92 Liu CS,Kong B,Xia HH,et al.VP22 enhanced intercellular trafficking of HSV thymidine kinase reduced the Level of ganciclovir needed to cause suicide cell death[J].J Gene Med,2001,3(2):145-152.
93 Phelan A,Elliott G,Intercellular delivery of functional p53 by the herpesvirus protein VP22[J].Nat Biotechnol, 1998,16(5):440-443.
94 Morris MC,Chaloin L,Heitz F,et al.Translocating peptides and proteins and their use for gene delivery[J].Curr Opin Biotechnol,2000,11:461 -466.
95 Niidome T,Ohmori N ,Ichinose A,et al.Binding of cationic alpha - helical peptides to plasmid DNA and their gene transfer abilities into cells[J].J Biol Chem,997,272(24): 15307- 15312.
96 Eguchi A,Akuta T,Okuyama H,et al.Protein transduction domain of HIV- 1 TAT protein promotes efficient delivery of DNA into mammalian cells[J].J Biol Chem,2001,276(28):26204-26210.
97 Astriab - Fisher A,Sergueev D,Fisher M,et al.Conjugates of antisense oligo nucleotides with the TAT and antennapedia cell-penetrating peptides: effects on celluLar uptake, binding to target sequences, and biologic actions[J].Pharm Res,2002,19(6):744-754.
98 Allinquant B,Hantraye P,Mailleux P,et al.Down regulation of amyloid precursor protein inhibits neurite outgrowth in vitro[J].J.Cell Biol,1995,128(5):919-927.
99 Derossi D,Chassaing G.Prochiantz.Trojan peptides: the penetratin system for intracellular delivery[J].Trends Cell Biol,1998,8(2):84-87.
100 Sandgren S,Cheng F,Belting M.Nuclear targeting of macro molecular poly anions by an HIV — TAT derived peptide.Role for cell - surface proteoglycans[J].J.Biol Chem,2002,277(41):38877-38883.
101 Rudolph C,Plank C,Lausier J,et al.Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells[J].J Biol Chem,2003,278(13):11411 - 11418.
102 Ignatovich IA,Dizhe EB,Pavlotskaya AV,et al.Complexes of pLasmid DNA with basic domain 47-57 of the HIV —1 TAT protein are transferred to mammalian cells by endocytosis — mediated pathways[J].J Biol Chem,2003,278(43): 42625-42636.
103 Tasciotti E,Zoppe M,Giacca M.Transcellular transfer of active HSV - 1 thymidine kinase mediated by an 11 - amino - acid peptide from HIV - 1 TAT[J].Cancer Gene Ther,2003,10(1):64-74.
104 Hashida H,Miyamoto M,Cho Y,et al.Fusion of HIV — 1 TAT protein transduction domain to poly—Lysine as a new DNA delivery tool[J].Br J Cancer,2004,90(6): 1252～1258.
105 Suzuki K,Murtuza B,Brand NJ,et al.Enhanced effect of myocardial gene transfection by VP22—mediated intercellular protein transport[J]. J MoL Cell Cardiol,2004,36(4):603～606.
106 Zavaglia D,Normand N,Brewis N,et al.VP22—mediated and Light—activated delivery of an anti—c—rafl antisense oligonucleotide improves its activity after intratumoral injection in nude mice[J].Mol Ther,2003,8(5): 840～845.
107 Zavaglia D,Favrot MC,Eymin B,et al.Inter cellular trafficking and enhanced in vivo antitumour activity of a non—virally delivered P27-VP22 fusion protein[J].GeneTher,2003,10(4):314～325.
108 Pooga M,Soomets U,Hallbrink M,et al.Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo[J].Nat Biotechnol,1998,16(9):857～861.
109 Cochet O,Kenigsberg M,Delumeau I,et al.Intracellular expression of an antibody fragment—neutralizing p21 ras promotes tumor regression[J].Cancer Res, 1998,58(6): 1170～1176.
110 Anderson DC,Nichols E,Manger R,et al.Tumor cell retention of antibody Fab fragments is enhanced by an attached HIV TAT protein derived peptide[J].Biochem Biophys Res Cornrnun,1993,194(2):876～884.
111 Stein S,Weiss A,Adermann K,et al.A disulfide conjugate between anti—tetanus antibodies and HIV (37-72)TAT neutralizes tetanus toxin inside chromaffin cells[J].FEBS Lett, 1999,458(3): 383～386.
112 Mie M,Takahashi F,Funabashi H,et al.Intracellular delivery of antibodies using TAT fusion protein A[J].Biochem Biophys Res Commun,2003,310(3):730～ 734.
113 Polyakov V,Sharma V,Dahlheimer JL,et al.Novel TAT—peptide chelates for direct transduction of technetium—99m and rhenium into human cells for imaging and radiotherapy[J].Bioconjug Chem,2000,11(6):762～771.
114 Bhorade R,Weissleder R,Nakakoshi T,et al.Macrocyclic chelators with paramagnetic cations are internalized into mammalian cells via a HIV—TAT derived membrane translocation peptide[J].Bioconjug Chem,2000,11(3): 301 - 305.
115 Bullok KE,Dyszlewski M,Prior JL,et al.Characterization of novel histidine- tagged TAT—peptide complexes dual —labeled with 99 mTc— tricarbonyl and fluorescein for scintigraphy and fluorescence microscopy[J].Bioconjug Chem,2002,13(6): 1226-1237.
116 Kaufman CL,Williams M,Ryle LM,et al.Superparamagnetic iron oxide particles transactivator protein- fluorescein isothiocyanate particle labeling for in vivo magnetic resonance imaging detection of cell migration: uptake and durability[J].Transplantation,2003,76(7): 1043- 1046.
117 Lewin M,Carlesso N,Tung CH,et al.TAT peptide - derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells[J].Nat Biotechnol,2000,18(4):410—414.
118 Liu J,Zhang Q,Remsen EE,et al.Nanostructured materials designed for cell binding and transduction[J].Biomacromolecules,2001,2(2):362—368.
119 Dodd CH,Hsu HC,Chu WJ,et al.Normal T-cell response and in vivo magnetic resonance imaging of T cellsloaded with HIV transactivator- peptide—derived superparamagnetic nanoparticlles[J].J Immunol Methods,2001,256(1- 2):89— 105.
120 Tkachenko AG,Xie H,Liu Y,et al.Cellular trajectories of peptide- modified gold particle complexes:comparison of nuclear localization signals and peptide transduction domains[J].Bioconjug Chem,2004,15(3):482—490.
121 Josephson L,Tung CH,Moore A,et al.High—efficiency intracellular magnetic labeling with novel superparamagnetic—TAT peptide conjugates[J].Bioconjug Chem, 1999,10(2): 186—191.
122 TorchiLin VP,Rammohan R,Weissig V,et al.TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low tern perature and in the presence of metabolic inhibitors[J].Proc Natl Acad,2001, 98(15):8786- 8791.
123 Levchenko TS,Rammohan R,Volodina N,et al.TAT peptide — mediated intracellular delivery ofliposomes[J].Methods Enzymol,2003,372:339-349.
124 Marty C,Meylan C,Schott H,et al.Enhanced heparan sulfate proteoglycan— mediated uptake of cell—penetrating peptide—modified liposomes[J]. Cell Mol Life Sci,2004,61(14):1785～1794.
125 Melan MA,Sluder G.Redistribution and differential extraction of soluble proteins in permeabilized cultured cells,Implications for immunofluorescence microscopy[J].J Cell Sci,1992,101(Pt4):731～743.
126 Pichon C,Monsigny M., Roche A.C.. IntracelluLar localization of oligonucleotides:influence of fixative protocols[J].Antisense Nucleic Acid Drug Dev,1999,9(1):89～93.
127 Lundberg M,Johansson M.Positively charged DNA—binding proteins cause apparent cellmembrane translocation[J].Biochem Biophys Res Commun,2002, 291(2):367～371.
128 Lundberg M,Johansson M.Is VP22 nuclear homing an artifact?[J].Nat Biotechnol,2001,19(8):713～714.
129 Richard JP,Melikov K,Vives E,et al.Cell—penetrating peptides: a reevaluation of the mechanism of cellular uptake[J]. J Biol Chem,2003,278(1):585～590.
130 Lundberg M,Wikstrom S,Johansson M.Cell surface adherence and endocytosis of protein transduction domains[J].Mol Ther,2003,8(1): 143～150.
131 Drin G,Cottin S,Blanc E,et al.Studies on the internalisation mechanism of cationic cell—penetrating peptides[J].J Biol Chem,2003,278(33):31192～31201.
132 Ho A,Schwarze SR,Mermelstein SJ,et al.Synthetic protein transduction domains:enhanced transduction potential in vitro and in vivo[J].Cancer Res,2001,61(2):474～477.
133 Richard JP,Melikov K,Vives E,et al.Cell—penetrating peptides:a reevaluation of the mechanism of cellular uptake[J].J Biol Chem,2003,278(1):585～590.
134 Lundberg M,Johansson M.Positively charged DNA—binding proteins cause apparent cell membrane translocation.Biochem[J].Biophys Res Commun, 2002,291 (2):367～371.
135 Lundberg M,Wikstrom S,Johansson M.Cell surface adherence and endocytosis of protein transduction domains[J].Mol Ther,2003, 8(1):143～150.
136 Koppelhus U,Awasthi SK, Zachar V,et al.Cell—dependent differential cellular uptake of PNA,peptides,and PNA -peptide conjugates[J].Antisense Nucleic Acid Drug Dev,2002,12(2):51-63.
137 Violini S,Sharma V,Prior JL,et al.Evidence for a plasma membrane mediated permeability barrier to tat basic domain in well - differentiated epithelial cells:Lack of correlation with heparan sulfate[J].Biochemistry 2002,41 (42): 12652-12661.
138 Kramer SD,Wunderli-Allenspach H.No entry for TAT44 —57 into liposomes and intact MDCK cells:novel approach to study membrane permeation of cell - penetrating peptides[J].Biochim Biophys Acta,2003,1609(2):161 - 169.
139 Falnes PO,Wesche J,Olsnes S.Ability of the Tat basic domain and VP22 to mediate cell binding, but not membrane translocation of the diphtheria toxin A— fragment[J].Biochemistry,2001,40(12):4349-4358.
140 Leifert JA,Harkins S,Whitton JL.Full -length proteins attached to the HIV tat protein transduction domain are neither transduced between cells,nor exhibit enhanced immunogenicity[J].Gene Ther,2002,9(21):1422—1428.
141 Fang B,Xu B,Koch P,et al.Intercellular trafficking of VP22 — GFP fusion proteins is not observed in cultured mammalian cells[J].Gene Ther, 1998,5:1420—1424.
142 Caron NJ,Torrente Y,Camirand G,et al.Intracellular delivery of a Tat —GFP fusion protein into muscle cells[J].Mol Ther,2001,3(3):310—318.
143 Bogoyevitch MA,Kendrick TS,Ng DC,et al.Taking the cell by stealth or storm? Protein transduction domains PTDs as versatile vectors for delivery[J].DNA Cell Biol,2002,21(12):879—894.
144 Hallbrink M,Floren A,Elmquist A,et al.Cargo delivery kinetics of cell — penetrating peptides[J].Biochim Biophys Acta,2001,1515(2): 101 — 109.
145 Mai JC,Shen H,Watkins SC,et al. Efficiency of protein transduction is cell type — dependent and is enhanced by dextran sulfate[J].J Biol Chem,2002,277 (33):30208—30218.
146 Tre'hin R,Cell penetrating peptides:uptake,permeation and metab olism in epithelial models[J].PhD Thesis,2004,5:15314.
147 Violini S,Sharma V,Prior JL,et al.Evidence for a plasma membrane- mediated permeability barrier to tat basic domain in well—differentiated epithelial cells:lack of correlation with heparan sulfate[J].Biochemistry,2002, 41(42):12652～12661.
148 Tre'hin R.Cell penetrating peptides:uptake,permeation and metabolism in epithelial models[J].PhD Thesis,2004,5:15314.
149 Elmquist A,Lindgren M,Bartfai T,et al.VE—cadherinderived cell—penetrat ing peptide,pVEC,with carrier functions[J].Exp Cell Res,2001,269(2):237～244.
150 Elmquist A,Langel U.In vitro uptake and stability study of pVEC and its all—D analog[J].Biol Chem,2003,384(3):387～393.
151 Soomets U,Lindgren M,Gallet X,et al.Deletion analogues of transportan[J]. Biochim Biophys Acta,2000,1467(1): 165～176.
152 Lindgren ME,Hallbrink MM,Elmquist AM,et al.Passageof cell—penetrating peptides across a human epithelial cell layer in vitro[J].Biochem J, 2004,377(1):69～76.
153 Bonavia R,Bajetto A,Barbero s,et al.HIV—1 Tat causes apoptotic death and calcium homeostasis aLterations in rat neurons[J].Biochem Biophys Res Commun,2001, 288(2):301～308.
154 Nath A,Psooy K,Martin C,et al.Identification of a human immunodeficiency virus type 1 Tat epitope that is neuroexcitatory and neurotoxic[J].J Virol, 1996,70(3): 1475～1480.
155 Jia H,Lohr M,Jezequel S,et al.Cysteine—rich and basic domain HIV—1 Tat peptides inhibit angiogenesis and induce endothelial cell apoptosis[J]. Biochem Biophys Res Commun,2001,283(2):469～479.
156 Bolton SJ,Jones DN,Darker JG,et al.Cellular uptake and spread of the cell— permeable peptide penetratin in adult rat brain[J].Eur J Neurosci,2000,12(8): 2847～2855.
157 Garcia—Echeverria C,Jiang L,Ramsey TM,et al.A new antennapedia—derived vector for intracellular delivery of exogenous compounds[J].Bioorg Med Chem Lett,2001,11(11):1363～1366.
158 Shahana S, Kampf C,Roomans GM.Effects of the cationic protein poly—L— arginine on airway epithelial cells in vitro[J].Mediators Inflamm,2002,11(3): 141～148.
159 Santana A,HysLop S,Antunes E,et al.Inflmmatory responses induced by poly—L —arginine in rat Lungs in vivo[J].Agents Actions, 1993,39(3—4):104～l10.
160 Koelle DM,Frank JM,Johnson ML,et al.Recognition of herpes simplex virus type 2 tegument proteins by CD4 T cells infiltrating human genital herpes lesions[J].J Virol,1998,72(9):7476～7483.
2 Simpkins N,Jankovic J.Neuroprotection in Parkinson Disease[J].Arch Intern Med,2003,163 (14):1650～1654.
3 Shumsky JS,Tobias CA,Tumolo M,et al.Delayed transplantation of fibroblasts genetically modified to secrete BDNF and NT—3 into a spinal cord injury site is associated with limited recovery of function[J].Experimental Neurology, 2003,184(1):114～130.
4 Von Bartheld CS,Johnson JE. Target- derived BDNF (brain- derived neurotrophic factor) is essential for the survival of developing neurons in the isthmo—optic nucleus[J].J Comp Neurol,2001,433(4):550～564.
5 Green M,Loewenstein PM.Autonomous functional domains of chemically synthesized human immunodeficiency virus Tat trans—activator protein[J].Cell, 1988,55(5): 1179～1188.
6 Frankel AD,Pabo Co. Cellular uptake of the Tat protein from human immuno deficiency virus[J].Cell,1988,55 (5) :1189～1193.
7 Sik Eum M,Soon Choung I,Zhen Li M,et al.HIV—1 tat mediated protein transduction of Cu,Z—superoxide dismutase into pancreaticβcells in vitro and in vivo[J].Free Radical Biology &Medicine,2004,37(3):339～349.
8 Becker—HapakM, McAllister SS, Dowdy SF. TAT mediated protein transduction into mammalian cells[J].Methods,2001,24 (3) :247～256.
9 Okada CY,Rechsteiner M.Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles[J].Cell,1982,29(1):33-41.
10 Chakrabarti R,Wylie DE,Schuster SM.Transfer of monoclonal antibodies into mammalian cells by electroporation[J].J Biol Chem, 1989,264(26): 15494 - 500.
11 Fawell S,Seery J,Daikh Y,et al.Tat-mediated delivery of heterologous proteins into cells[J].Proc. Natl Acad Sci USA,1994,91(2):664-668.
12 Vives E,Brodin P,Lebieu B.A truncated HIV- 1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus[J].J Biol Chem, 1997,272(25): 16010 - 16017.
13 Steven R S,ALan Ho,Adamina V et al .In vivo protein transduction : delivery of a biologically active protein into the mouse[J].Science, 1999,285(5433): 1569- 1572.
14 Futaki S,Suzuki T,Ohashi et al.Arginine-rich peptides.An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery[J].J Biol Chem,2001;276(8):5836-5840.
15 Brooks H,Lebleu B,Vives E.Tat peptide-mediated cellular delivery: back to basics[J].Adv Drug Deliv Rev, 2005;57(4):559-577.
16 Trehin R,Merkle HP.Chances and pitfalls of cell penetrating peptides for cellular drug delivery[J]. Eur J Pharm Biopharm,2004,58(2):209-223.
17 Rothbard JB,Jessop TC,Lewis RS, et al. Role of membrane potential and hydrogen bonding in the mechanism of translocation of guanidinium - rich peptides into cells[J].J Am Chem Soc,2004,126(31):9506-9507.
18 Schwarze.SR,Ho A,Vocero - Akbani A,et al.In Vivo Protein Transduction: Delivery of a Biologically Active Protein into the Mouse science, 1999, 285:1569-1572.
19 Jin LH,Bahn JH,Eum WS et al .Transduction of human catalase mediated by an HIV- 1 TAT protein basic domain and arginine-rich peptides into mammalian cells. Free Radic Biol Med,2001,31(11): 1509-1519.
1 Shumsky JS,Tobias CA,Tumolo M,et al.Delayed transplantation of fibroblasts genetically modified to secrete BDNF and NT—3 into a spinal cord injury site isassociated with limited recovery of function[J].Experimental Neurology,2003, 184(1):114～130.
2 Von Barthelld CS,Johnson JE.Target—derived BDNF (brain—derived neurotrophic factor) is essential for the survival of developing neurons in the isthmo—optic nucleus[J].JComp Neurol,2001,433(4):550～564.
3 Shults CW,Kinker T.BDNF attenuates the effects of intranstraital injection of 6 —hydroxydopamine[J].Neurorepor, 199,6(8): 1109.
4 Iskandrian AS. Adenosine myocardial perfusion imaging[J]. J Nuel Med ,1994, 35(4): 734.
5 Minichiello L,Calella A,Medina D,et al.Mechanism of TrkB—mediated hippocampal long—term potentiation[J].Neuron,2002,36(1): 121.
6 Green M,Loewenstein PM.Autonomous functional domains of chemically synthesized human immunodeficiency virus Tat trans—activator protein[J]. Cell,1988, 55(5):1179～1188.
7 Frankel AD,Pabo Co.Cellular uptake of the Tat protein from human immunodeficiency virus[J].Cell, 1988,55(5): 1189～1193.
8 Vives E,Brodin P,Lebieu B. A truncated HIV—1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus[J].J Biol Chem, 1997,272(25): 16010～16017.
9 Schwarze SR,Ho A,Vocero--Akbani A,et al.In Vivo Protein Transduction: Delivery of a Biologically Active Protein into the Mouse[J].science,1999, 285(5433): 1569～1572.
10 Becker—HapakM, McALLister SS,Dowdy SF. TAT mediated protein transduction into mammalian cells[J].Methods,2001,24 (3) :247～256.
11 Barde Y A, Edgar D, Thoenen H, Purification of new neurotrophicfactor from brain [J] EMBO J,1982,1(5):549v553.
12 Ivanova T, Beyer C, Preo and postnatal expression of brain derived neurotro— phic foctor mRNA/protein and tyrosine protein kinage receptor B mRNA in the mouse hippocampos[J]. Neurosci lett,2001,307(1):21—24.
13 李力燕，王廷华等．脑源性神经营养因子在成年猴脑的分布．神经解剖学杂志，2002(1)：67—70．
14 Zhou J,Bradford HF, Stern GM,et al.The response of human and rat fetal ventral mesencephalon in culture to the brain—derived neurotrophic factor treatment[J]. Brain Res, 1994,656(1): 147～156.
15 Studer L, Spenger C, Seiler RW, et al. Effects of brain—derived neurotrophic factor on neuronal structure of dopaminergic neurons in dissociated cultures of human fetal mesencephalon[J].Exp Brain Res, 1996,108(2):328～336.
16 Michel PP, Ruberg M, Agid Y. Rescue of mesencephalic dopamine neurons by anticancer drug cytosine arabinoside[J].J Neurochem, 1997,69(4): 1499～1507.
17 Hoglinger GU, Sautter J, Meyer M, et al. Rat fetal ventral mesencephalon grown as solid tissue cultures: influence of culture time and BDNF treatment on dopamine neuron survival and function[J].Brain Res, 1998,813:313～322.
18 Spenger C, Hyman C, Studer L,et al. Effects of BDNF on dopaminergic, serotonergic, and GABAergic neurons in cultures of human fetal ventral mesencephalon[J].Exp Neurol, 1995,133(2):50～63.
19 Beck KD,Knusel B,Hefti F.The nature of the trophic action of brain—derived neurotrophic factor, des (1—3)--insulin--like growth factor--1, and basic fibroblast growth factor on mesencephalic dopaminergic neurons developing in culture[J].Neuroscience,1993,52(4):855～866.
20 Kontkanen O, Castren E. Trophic effects of selegiline on cultured dopaminergic neurons[J].Brain Res, 1999,829(1—2): 190～192.
21 Skaper SD,Negro A,Facci L,et al.Brain—derived neurotrophic factor selectiveLy rescues mesencephalic dopaminergic neurons from 2,4,5- trihydroxyp henylalanine—induced injury[J].J Neurosci Res,1993,34(4):478～487.
22 Spina MB, Squinto SP, Miller J,et al. Brain—derived neurotrophie factor protects dopamine neurons against 6—hydroxydopamine and N—methyl—4— phenylpyridinium ion toxicity: involvement of the glutathione system. J Neurochem[J], 1992,59(1):99～106.
23 Menei P , Whittemor S R .Schwann cells genetically modified to secrete human BDNF promote enhanced axonal regrowth across transected adult rat spinal cord[J].European Journal of Neuroscience, 1998,10(2):607～621.
24 Rubio F, Kokaia Z,Arco A, et al.BDNF gene transfer to the mammalian brain using CNS—derived neural precursors[J].Gene Ther, 1999, 6(11): 1851～1866.
25 Maisonpierre PC,Belluscio L,Squinto S,et al.Neurotrophin3:A neurotrophic factor related to NGF and BDNF[J].Science, 1990,247:1446～1451
26 Nagahara H, Snyder E.L, Snyder EL, et al.Transduction of full—length Tat fusion proteins into mammalian cells:TAT—p27Kipl induces cell migration[J].Nature Medicine. 1998,4(12): 1449～1452.
27 Berger B,Diporzio U,Daguet MC,et al.Long term development of mesencephalic dopaminergic and non—dopaminergic neurons in organotypic slice cultures of the rat ventral mesencephalon[J].ExpBrain Res, 1995,106(2):205～214.
28 Jia XJ,Gong SL, Liu SZ,et al.Dopaminergic neuron as the cell model for Parkinson's disease[J].J Chin J Neuromed,2004,3(5):382～386.
29 苏雅茹，蒋雨平．6—多巴胺在帕金森病发病中的作用[J]．中国临床神经科学．2002，10(3)：312～314．
30 Nass R,Hall DH,Miller DM,et al.Neurotoxin—induced degeneration of dopamine neurons in Caenorhabditis elegans[J].Neurobiology,2002,99(5): 3264～3269.
31 马敬红，胡国华，董丽华，等．6—羟基多巴在帕金森病发病机制中的作用[J]．吉林大学学报：医学版，2005，31(3)：355～357．
32 Zhang J, Liu X.Study on tetrazolium salt colorimetric assay for growth and survival of bacteria[J].Wei Sheng Yah Jiu,2002,31(5):361～363.
1 徐德隆，陈生弟，刘振国，主编．帕金森病临床新技术[J]．北京，人民军医出版社，2002年，第1版，p49～50．
2 Simpkins N,Jankovic J.Neuroprotection in Parkinson Disease[J].Arch Intern Med, 2003,163(14):1650～1654.
3 Green M,Loewenstein PM.Autonomous functional domains of chemically Synthesized human immunodeficiency virus Tat trans — activator protein[J].Cell,1988,55(5): 1179～1188.
4 Frankel AD,Pabo CO.Cellular uptake of the Tat protein from human immunodeficiency virus[J].Cell, 1988,55:1189～1193.
5 Fawell S,Seery J,Daikh Y, et al. Tat—mediated delivery of heterologous proteins into celLs[J].Proc. Natl Acad Sci USA, 1994,91 (2):664～8.
6 Perry TL, Ito M. Nigrostriatal dopam inergicneurons remain undamaged in rats given high doses of L — DOPA and caxbidopa chronically[J]. Neurochem, 1984,43(3):990～3.
7 包新民，舒斯云,主编．人鼠脑立体定位图谱[M]．北京人民卫生出版社，1991年，第1版,49～58．
8 9 Daly TM,Okuyama T, Vogler C,et al.Neonatal intramuscular injection with recombinant adeno — associated virus results in prolonged beta—glucuronidase expression in situ and correction of liver pathology in mucopolysac—charidosis type Ⅶ mice[J].Hum.Gene Ther, 1999,10(1):85～94.
9 盛树力，临床神经科学前沿[M]．北京：北京大学医学出版社,2003,20(7)：5411～5417．
10 Hattori N,Machida Y, Noda K. Pathogenesis of Parkinson's disease: a common pathway between alpha—synuclein and parkin and the mechanism of lewy bodies formation[J].Rinsho Shinkeigaku,2005,45(11):905～907.
11 Backlund EO, Granberg PO, Hamberger B, et al. Transplantation of a adrenal medullary tissue to striatum in Parkinsonism first clinical trials[J].Neurosurg, 1985,62(2): 169～173
12 Widner H,Tetrud J,Rehncrona S,et al.Bilateral fetal mesencephalic grating in two patients with parkinsonism induced by 1—methyl—4—phenyl—1,2,3,6—tetr ahydropyridine(MPTP)[J].Nat Eng J Med, 1992,327(22): 1556～1563
13 Lindvall O.Update on fetal transplantation:the Swedish experience [J].Mov.Disord, 1998,13:83～87.
14 Li JY, Brundin P. Grafting dopamine neurons in Parkinson's disease: Do stem cells have a role in the future? [J].Neurochem,2003,85(5): 13～15.
15 Mark S, Meena J,Roger A.The cellular repair of the brain in Parkinson's disease —past,present and future[J].Transplant Immunology,2004,12(3—4):321～342
16 陈生弟主编．帕金森病临床新技术[M]．人民军医出版社．2002年第1版．140～223．
17 Ronald D,Arjan B,Jos P.Modeling Parkinson's disease in rats:An evaluation of 6 —OHDA lesions of the nigrostriatalpathway[J].Experimental Neurology, 2002,175(2):303～317.
18 赵焕英，苏月，杨秋慧等．6—羟基多巴胺纹状体内注射制作大鼠帕金森病模型的研究[J]．中国组织化学与细胞化学杂志，2003，12(1)：16～21
19 Darwin J,Prockop,Carl A,et al.One strategy for cell and gene therapy:Harnessing the power of adult stem cells to repair tissues[J].PNAS September,2003, 100:11917～11923
20 Prockop DJ.Marrow Stromal Cells as Stem Cells for Momhematopoietic tis sues[J].Science, 1997,276:71～74
21 Deari C,Dell'Accio F, Vandenabeele F, et al.Skeletal muscle repair by a—dolt human mesenchymal stem cells from synovial membrane[J].Cell Biol,2003, 160(6):909～9184
22 Sanchez Ramos J,Song S,Cardozo Pelaez F, et al.Adult bone marrow stromal cells diferentiate into neural cells in vitro[J].Exp Neurol,2000,164(2):247～256
23 Woodbury D,Schwarz EJ,Prockop DJ,et al.Adult rat and human bone marrow stromal cells diferentiate into neurons[J].Neurosic Res,2000,61 (4):364～370
24 Azizi SA,Stokes D,Augelli B,et al. Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats similarities to astrocyte grafts[J]. Proc Natl Acad Sci USA,1998,95(7):3908～3913
25 Mezey E,Chandross KJ,Harta G, et al.Turning blood into brain:cells bearing neuronal antigens generated in vivo from bone marrow[J].Science,2000, 290(5497): 1779～1782
27 Meakin SO,Shooter EM.The growth factor family of receptors[J].Trends Neurosci, 1992,15:323～331
27 Orlic D,Kajstura J,Chimenti S,et al.Bone marrow cells regenerate infarcted myocardium[J].Nature,2001.410(6829):701～705
28 Murphy JM,KavaLkovitch KW, Fink D, et al.Regeneration of meniseal tissue and protection of articult cartilge by onjection of mesenchymalstem cells[J]. Osteoarthritis Cartilage,2000,8:25～29
29 Chen J,Li Y, Wang L,et al. Therapeutic benefit of intravenous administration of bone stromal cells after cerebral ischemia in rata[J].Stroke,2001,32(4):1005～1011
1 Varga CM,Wickham TJ,Lauffenburger DA.Receptor—mediated targeting of gene delivery vectors:insights from molecular mechanisms for improved vehicle design[J].Biotechnol,2000,70(6):593～605.
2 Chakrabarti R, Wylie DE,. Schuster SM.Targeting of gene delivery vectors:insights from molecular mechanisms for improved vehicle design[J].J. Biol. Chem, 1989,264:15494～15500.
3 Arnheiter H, Haller O.Antiviral state against influenza virus neutralized by microinjection of antibodies to interferon—induced Mx proteins[J].EMBO J, 1988,7(5): 1315～1320.
4 Green M,Loewenstein PM.Autonomous functional domains of chemically synthesized human immunodeficiencyvirus TAT trans—activator protein[J].Cell,1988,55 (6) : 1179～1188.
5 Frankel AD,Pabo CO.Cellular uptake of the TAT protein from human immuno deficiency virus[J].Cell,1988,55 (6) :1189～1193.
6 Joliot A, Pernelle C, Deagostini H, et al.Antennapedia homeo box peptide regulates neural morphogenesis[J].Proc Natl Acad Sci, 1991,88(5): 1864～1868.
7 Elliott G., O'Har P.e. Intercellular trafficking and protein delivery by a herpesvirus structural protein[J].Cell. 1997, 88(2):223～233.
8 Lindgren M, Hallbrink M,Prochiantz A, et al.Cell — penetrating peptides[J].Trends Pharmacol.Sci,2000,21 (3):99～103.
9 Hallbrink M,FLoren A,Elmquist A,et al.Cargo delivery kinetics of cell— penetrating peptides[J].Biochim Biophys Acta,2001,1515(2): 101～109.
10 Frankel AD,Bredt DS,Pabo CO.TAT protein from human immuno—deficiency virus forms a metal—linked dimer[J].Science, 1988,240:70～73.
11 Frankel AD, Chen L, Cotter R J, et al. Dimerization of the TAT protein from human immunodeficiency virus:a cysteine—rich peptide mimics the normal metallinkeddimer interface[J].Proc Natl Acad Sci, 1988,85(17):6297～6300.
12 Ruben S, Perkins A, Purcell R, et al.Structural and functional characterization of human immuno deficiency virus TAT protein[J].J Virol, 1989,63(1): 1～8.
13 Siomi H, Shida H, Maki M, et al. Effects of a highly basic region of human immunodeficiency virus TAT protein on nucleolar localization[J] .J Virol, 1990,64(4): 1803-1807.
14 Frankel AD, Biancalana S, Hudson D, Activity of synthetic peptides from the TAT protein of human immunodeficiency virus type l[J].Proc Natl Acad Sci,1989,86(19):397-401.
15 Mann DA, Frankel AD.Endocytosis and targeting of exogenous HIV-1 TAT Protein[J].EMBO J,1991,10(7):1733-1739.
16 Loret E.P,Vives E,Ho PS, et al.Activating region of HIV-1 TAT protein vacuum UV circular dichroism and energy minimization[J].Bioch emistry,1991,30(24):6013-6023.
17 Ruben S,Perkins A,Purcel Rl,et al.Structural and functional characterization of human immuno deficiency virus TAT protein[J].J Virol, 1989,63(1):1 -8.
18 Schwarze SR, Hruska KA, Dowd SF.Protein transduction unrestricted delivery into all cells?[J].Trends Cell Biol, 2000,10(7):290-295.
19 Gehring WJ,Affolter M, Burglin T. Homeodomain proteins[J].Annu Rev Biochem, 1994,63:487-526.
20 Derossi D, Jolio AH, Chassaing G, et al. The third helix of the Antennapedia homeodomain translocates through biological membranes[J].J Biol Chem, 1994,269(14): 10444- 10450.
21 Elliott G, O'Hare P. Herpes simplex virus type 1 tegument protein VP22 induces the stabilization and hyperacetylation of microtubules[J].J Virol, 1998, 72(8):6448-6455.
22 Aints A,Guven H,Gahrton G,et al.Mapping of herpes simplex virus-1 VP22 functional domains for inter - and sub cellular protein targeting[J].Gene Ther,2001,8(14):1051-1056.
23 Pooga M,Hallbrink M,Zorko M,et al.Cell penetration by transportan[J]. FASEBJ,1998,12(1):67-77.
24 Oehlk JE,Scheller A, Wiesner B,et al.Cellular uptake of an alpha -helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically[J].Biochim Biophys Acta,1998,1414(1 - 2):127-139.
25 Lin YZ, Yao SY. Inhibition of nuclear translocation of trans cription factor NFkappaB by a synthetic peptide containing a cell memb rane permeable motif and nuclear loca - lization sequence[J] . J Biol Chem, 1995,270(24): 14255 - 14258.
26 Oess S, Hildt E.Novel cell permeable motif derived from the PreS2-domain of hepatitis-B virus surface antigens[J].Gene Ther,2000,7(9):750-758.
27 Mi Z,Mai J,Lu X,et al.Characterization of a class of cationic peptides able to faciliTATe efficientprotein transduction in vitro and in vivo[J].Molec Ther, 2000,2(4):339-347.
28 Gorodetsky R,Levdansky L, Vexler A,et al.Liposome transduction into cells enhanced by haptotactic peptides Haptides homologous to fibrinogen C - termini[J]. J Control Release,2004,95(3):477-488.
29 Derossi D, Joliot AH, Chassaing G, et al.The third helix of the antenna pedia homeodomain translocates through biological membranes[J].J Biol Chem, 1994,269:10444-10450.
30 Derossi D,Calvet S,Trembleau A,et al.Cell internalization of the third helix of the antennapedia homeodomain is receptor - independent[J].J Biol Chem, 1996,271(30):18188-18193.
31 Elliott G,O'Hare P,Intercellular trafficking and protein delivery by a herpesvirus structural protein[J].Cell,1997,88(2):223-233.
32 Vives E,Brodin P,Lebleu B.A truncated HIV - 1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus[J].J Biol Chem,1997,272(25):16010-16017.
33 Vogel BE, Lee SJ, HiLdebrand A.A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin[J].J Cell Biol,1993,121(2):461-468.
34 Fender PA.The design, synthesis,and evaluation of molecule:that enable or enhance cellular uptake :peptoid molecular transporters[J].Proc Natl Acad,2000,97(24):l 3003-13008.
35 Vives E,Grannier C,Prevot P,et al.Structure activity relationship study of the plasma membrane translacating potential of a short peptide from HIV-1 Tat protein[J].Lett Peptide, 1997,4:429～436.
36 Futaki S,Suzuki T, Ohashi W, et al.Arginine—rich Peptides:An abundant source of membrane—permeable peptides having potential as carriers for intracellular protein delivery[J].J Biol Chem,2001,276(8):5836～5840.
37 Suzuki T, Futaki S, Niva M, et al.Possible existence of common internalization mechanism among arginine—rich peptide[J].J Biol Chem,2002,277(4):2437～3443.
38 Deros D,Joliot AH, Chassaing G, et al.The third helix of the Antennapedia homeodomain translocate:through biological membrane[J].J Biol Chem, 1994,269:10444～10450
39 Silhol M, Tyagi M, Giacca M, et al.Different mechanisms for cellular internalization of the HIV—1 Tat—derived cell penetrating peptide and recombinant proteins fused to Tat[J].Eur J Biochem,2002,269(2):494～501
40 Schwarze SR, Hruska KA,Dowdy SF.Protein transduction: unrestricted delivery into all cells?[J].Trends Cell Biol,2000,10(7):290～295.
41 Tyagi M, Rusnati M, Presta M, et al.Cell surface heparan sulfate nternalization of HIV—1 tat requiresproteoglycan[J].J Biol Chem,2001,276:3254～3261.
42 Belting M.Heparan sulfate proteoglycan as a plasma membrane carrier[J].Trends Biochem Sci,2003,28(3): 145～151.
43 Silhol M, Tyag M, Giacca M, et al. Different mechanisms for cellular internalization of the HIV—1 Tat—derived cell penetrating peptide andrecombinant proteins fused to Tat[J].Eur J Biochem,2002,269:494～501.
44 Gehring W J, Muller M,Affolter M,et al The structure of the homeodomain and its functional implications[J].Trends Genet, 1990,6(10):323～329.
45 Prochiantz A.Getting hydrophilic compounds into cells:Lessons from homeopeptides [J].Opin. Neurobiol,1996,6(5):629～634.
46 Prochiantz A.Homeodomain—derived peptides:In and out of the cells[J].Ann N Y Acad Sci,1999,886:172～179.
47 Le Roux I, Duharcourt S, Volovitch M, et al. Promoter—specific regulation of gene expression by an exogenouslyadded homedomain that promotes neurite growth[J]. Fed Eur Biochem Soc Lett,1995,368(2):311～314.
48 Drin G, Mazel M, CLair P, et al. Temsamani, Physico-chemical requirements for cellular uptake of pAntp peptide.Role of Lipid - binding affinity[J].Eur J.Biochem,2001,268(5): 1304- 1314.
49 Joliot AH,Triller A,Volovitch M,et al.Lpha-2,8-Polysialic acid is the neuronal surface receptor of antennapedia homeobox peptide[J].New Biol. 1991, 3(11):1121 - 1134.
50 Perez F,Joliot A,Bloch E,et al.Antennapedia homeobox as a signal for the cellularinternalization and nuclear addressing of a small exogenous peptide[J].J Cell Sci,1992,102(Pt4):717-722.
51 Derossi D,Chassaing G, Prochiantz A,Trojan peptides: the penetratin system for intracellular delivery[J].Trends Cell Biol,1998,8:84-87.
52 Berlose JP,Convert O,Derossi D,et al.Chassaing,Conformational and associative behaviours of the third helix of antennapedia homeodomain in membrane- mimetic environments[J].Eur J Biochem, 1996,242(2):372-386.
53 Thoren PE,Persson D,Karlsson M,et al.The antennapedia peptide penetratin translocates across lipid bilayers-the first directobservation[J].Fed Eur Biochem Soc Lett,2000,482(3):265-268.
54 Drin G,Cottin S,Blanc E,et al.Temsamani, Studies on the internalisation mechanism of cationic cell - penetrating peptides[J].J Biol Chem,2003,278: 31192-31201.
55 Elliott GD,Meredith DM.The herpes simplex virus type 1 tegument protein VP22 is encoded by gene[J].J Gen Virol, 1992,73(Pt3):723-726.
56 Kuchler K.Unusual routes of protein secretion: the easy way out[J].Trends Cell Biol,1993,3(12):421-426.
57 Mann DA,Frankel AD.Endocytosis and targeting of exogenous HIV - 1 Tat protein[J].Eur Mol Biol Org, 1991,10(7): 1733 -1739.
58 Frankel AD,Pabo CO.Cellular uptake of the tat protein from human immunodeficiency virus[J].Cell, 1988,55:1189-1193.
59 Rusnati M,Tulipano G,Urbinati C,et al.The basic domain in HIV-1 Tat protein as a target for polysulfonated heparin - mimicking extracellular Tat antagonists[J].J Biol Chem, 1998,273(26): 16027-16037.
60 Brake DA,Debouck C, Biesecker G.Identification of an Arg—GLy—Asp (RGD) cell adhesion site in human immunodeficiency virus typel transacfivation protein tat[J].J Cell Biol, 1990,111 (3): 1275～1281.
61 Ferrari A,Pellegrini V,Arcangeli C,et al.Caveolae—mediated interna lization of extracellular HIV—1tat fusion proteins visualized in real time[J].Mol Ther, 2003,8(2):284～294.
62 Richard JP,Melikov K,Vives E,et al.Cell—penetrating peptides:are—evaluation of the mechanism of cellular uptake[J].J Biol Chem,2003,278(1):585～590.
63 Koppelhus U,Awasthi SK,Zachar V,et al.Cell—dependent differential cellular uptake of PNA,peptides, and PNA—peptide conjugates[J].Antisense Nucleic Acid Drug,2002,12(2):51～63.
64 Lundberg M, Wikstrom S, Johansson M.Cell surface adherence and endocytosis of protein transduction domains[J].Mol Ther,2003,8(1): 143～150.
65 Console S,Marty C,Garcia C—Echeverria, et al.Antennapedia and HIV TAT protein transduction domains promote endocytosis of high Mr cargo upon binding to cell surface gLycosaminoglycans[J].J Biol Chem,2003,278(37): 35109～35114.
66 Sandgren S,Cheng F,Belting M.Nuclear targeting of macro molecular polyanions by an HIV—Tat derived peptide. Role for cellsurface proteoglycans[J].J Biol Chem,2002,277(41):38877～38883.
67 Hallbrink M,Floren A,Elmquist A,et al.Cargo delivery kinetics of cell— penetrating peptides[J].Biochim Biophys Acta,2001,1515(2): 101～109.
68 Rousselle C, Smirnova M, Clair P,et al.Enhanced delivery of doxorubicin into the brain via a peptide—vector—mediatedstrategy: saturation kinetics and specificity[J].J Pharmacol Exp Ther,2001,296(1): 124～131.
69 Fawell S,Seery J,Daikh Y,et al.TAT—mediated delivery of heterologous proteins into cells[J].Proc Natl Acad Sci, 1994,91(2):664～668.
70 Moy P,Daikh Y,Pepinsky B,et al.TAT—mediated protein delivery can facilitate MHC class I presentation of antigens[J].Mol Biotechnol, 1996,6(2): 105～13.
71 Kim DT,Mitchell DJ,Brockstedt DG,et al.Introduction of soluble proteins into the MHC class I pathway by conjugation to an HIV TAT peptide[J].J Immunol, 1997,159(4): 1666～1668.
72 Schwarze SR,Ho A,Vocero—Akbani A, et al.In vivo protein transduction:delivery of a biologically active protein into the mouse[J].Science,1999,285(5433): 1569～1572.
73 Caron NJ,Torrente Y,Camirand G,et al.Intracellular delivery of a TAT-eGFP fusion protein into musclecells[J].Molec Ther,2001,3(3):310～318.
74 Vocero— Akbani A,Lissy N,Dowdy SF.Transduction of full—length TAT fusion proteins directly into mammalian cells:analysis of T cell receptor activation—induced cell death[J].Methods Enzymol,2000,322:508～521.
75 Becker—Hapak M,Mcallister SS,Dowdy SF.TAT mediated protein trans duction into mammalian cells[J].Methods,2001,24(3):247～256.
76 Nagahara H,Vocero—Akbani AM,Snyder EL,et al.Transduction of full—Length TAT fusion proteins intomammalian cells:TAT—p27Kip1 induces cell migration[J].Nat Med,1998,4(12):1449～1452.
77 Embury J,Klein D,Pileggi A,et al.Proteins linked to a protein transduction domain efficiently transduce pancreatic islets[J].Diabetes,2001,50(8): 1706～ 1713.
78 Dolgilevich S,Zaidi N,Song J,et al.Transduction of TAT fusion proteins into osteoclasts and osteoblasts[J].Biochem Biophys Res Commun,2002,299(3): 505～509.
79 Kwon HY,Eum WS,Jang HW,et al.Transduction of Cu,Zn—superoxide dismutase mediated by an HIV—1 TAT protein basic domain into mammalian cells[J].FEBS Lett,2000,485(2—3): 163～167.
80 Jin LH,Bahn JH,Eum WS,et al.Transduction of human catalase mediated by an HIV—1 TAT protein basic domain and arginine—rich peptides into mamma lian cells[J].FreeRadicBiol Med,2001,31(11):1509～1519.
81 Yang Y,Ma J,Song Z,et al.HIV—1 TAT—mediated protein trans duction and subcellular Localization using novel expression vectors[J].FEBS Lett,2002,532 (1-2):36～44.
82 Wheeler DS,Dunsmore KE,Wong HR.Intracellular delivery of HSP70 using HIV —1 TAT protein transduction domain.Biochem[J] .Biophys Res Commun, 2003,301(1):54-59.
83 Yoon HY,Lee SH,Cho SW,et al.TAT-mediated delivery of human glutamate dehydrogenase into PC 12 cells[J].Neurochem,2002,41(1):37-42.
84 Shibagaki N,Udey MC.Dendritic cells transduced with protein antigens induce cytotoxic lymphocytes and elicit antitumor immunity[J].J Immunol, 2002,168(5):2393-2401.
85 Wang HY,Fu T,Wang G,et al.Induction of CD4(+)T cell-dependent antitumor immunity by TAT-mediated tumor antigen delivery into dendritic cells[J].J Clin Invest,2002,109(11):1463- 1470.
86 Kabouridis PS,Hasan M,Newson J,et al.Inhibition of NF-kappa B activity by a membrane transducing mutant of I kappa B alpha[J].J Immunol,2002,169 (5):587-2593.
87 Gustafsson AB,Sayen MR,Williams SD,et al.TAT protein transduction into isolated perfused hearts: TAT— apoptosis repressor with caspase recruit ment domain is cardio protective[J].Circulation,2002,106(6):735-739.
88 Cao G,Pei W,Ge H,et al.In vivo delivery of a Bcl—x1 fusion protein contain ing the TAT protein transduction domain protects against ischemic brain injury and neuronal apoptosis[J].J Neurosci,2002,22(13):5423-5431.
89 Asoh S,Ohsawa I,Mori T,et al.Protection against ischemic brain injury by protein therapeutics[J].Proc Natl Acad Sci,2002,99(26):17107- 17112.
90 Roeder GE,Parish JL,Stem PL,et al.Herpessimplex virus VP22-human papillomavirus E2 fusion proteins produced in mammalian or bacterial cells enter mammalian cells and induce apoptotic cell death[J].Biotechnol Appl Biochem,2004,40(Pt2): 157-165.
91 Derer W,Easwaran HP,Leonhardt H,et al.A novel approach to induce cell cycle reentry in terminally differentiated muscle cells[J].FASEB J,2002,16(1): 132- 133.
92 Liu CS,Kong B,Xia HH,et al.VP22 enhanced intercellular trafficking of HSV thymidine kinase reduced the Level of ganciclovir needed to cause suicide cell death[J].J Gene Med,2001,3(2):145-152.
93 Phelan A,Elliott G,Intercellular delivery of functional p53 by the herpesvirus protein VP22[J].Nat Biotechnol, 1998,16(5):440-443.
94 Morris MC,Chaloin L,Heitz F,et al.Translocating peptides and proteins and their use for gene delivery[J].Curr Opin Biotechnol,2000,11:461 -466.
95 Niidome T,Ohmori N ,Ichinose A,et al.Binding of cationic alpha - helical peptides to plasmid DNA and their gene transfer abilities into cells[J].J Biol Chem,997,272(24): 15307- 15312.
96 Eguchi A,Akuta T,Okuyama H,et al.Protein transduction domain of HIV- 1 TAT protein promotes efficient delivery of DNA into mammalian cells[J].J Biol Chem,2001,276(28):26204-26210.
97 Astriab - Fisher A,Sergueev D,Fisher M,et al.Conjugates of antisense oligo nucleotides with the TAT and antennapedia cell-penetrating peptides: effects on celluLar uptake, binding to target sequences, and biologic actions[J].Pharm Res,2002,19(6):744-754.
98 Allinquant B,Hantraye P,Mailleux P,et al.Down regulation of amyloid precursor protein inhibits neurite outgrowth in vitro[J].J.Cell Biol,1995,128(5):919-927.
99 Derossi D,Chassaing G.Prochiantz.Trojan peptides: the penetratin system for intracellular delivery[J].Trends Cell Biol,1998,8(2):84-87.
100 Sandgren S,Cheng F,Belting M.Nuclear targeting of macro molecular poly anions by an HIV — TAT derived peptide.Role for cell - surface proteoglycans[J].J.Biol Chem,2002,277(41):38877-38883.
101 Rudolph C,Plank C,Lausier J,et al.Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells[J].J Biol Chem,2003,278(13):11411 - 11418.
102 Ignatovich IA,Dizhe EB,Pavlotskaya AV,et al.Complexes of pLasmid DNA with basic domain 47-57 of the HIV —1 TAT protein are transferred to mammalian cells by endocytosis — mediated pathways[J].J Biol Chem,2003,278(43): 42625-42636.
103 Tasciotti E,Zoppe M,Giacca M.Transcellular transfer of active HSV - 1 thymidine kinase mediated by an 11 - amino - acid peptide from HIV - 1 TAT[J].Cancer Gene Ther,2003,10(1):64-74.
104 Hashida H,Miyamoto M,Cho Y,et al.Fusion of HIV — 1 TAT protein transduction domain to poly—Lysine as a new DNA delivery tool[J].Br J Cancer,2004,90(6): 1252～1258.
105 Suzuki K,Murtuza B,Brand NJ,et al.Enhanced effect of myocardial gene transfection by VP22—mediated intercellular protein transport[J]. J MoL Cell Cardiol,2004,36(4):603～606.
106 Zavaglia D,Normand N,Brewis N,et al.VP22—mediated and Light—activated delivery of an anti—c—rafl antisense oligonucleotide improves its activity after intratumoral injection in nude mice[J].Mol Ther,2003,8(5): 840～845.
107 Zavaglia D,Favrot MC,Eymin B,et al.Inter cellular trafficking and enhanced in vivo antitumour activity of a non—virally delivered P27-VP22 fusion protein[J].GeneTher,2003,10(4):314～325.
108 Pooga M,Soomets U,Hallbrink M,et al.Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo[J].Nat Biotechnol,1998,16(9):857～861.
109 Cochet O,Kenigsberg M,Delumeau I,et al.Intracellular expression of an antibody fragment—neutralizing p21 ras promotes tumor regression[J].Cancer Res, 1998,58(6): 1170～1176.
110 Anderson DC,Nichols E,Manger R,et al.Tumor cell retention of antibody Fab fragments is enhanced by an attached HIV TAT protein derived peptide[J].Biochem Biophys Res Cornrnun,1993,194(2):876～884.
111 Stein S,Weiss A,Adermann K,et al.A disulfide conjugate between anti—tetanus antibodies and HIV (37-72)TAT neutralizes tetanus toxin inside chromaffin cells[J].FEBS Lett, 1999,458(3): 383～386.
112 Mie M,Takahashi F,Funabashi H,et al.Intracellular delivery of antibodies using TAT fusion protein A[J].Biochem Biophys Res Commun,2003,310(3):730～ 734.
113 Polyakov V,Sharma V,Dahlheimer JL,et al.Novel TAT—peptide chelates for direct transduction of technetium—99m and rhenium into human cells for imaging and radiotherapy[J].Bioconjug Chem,2000,11(6):762～771.
114 Bhorade R,Weissleder R,Nakakoshi T,et al.Macrocyclic chelators with paramagnetic cations are internalized into mammalian cells via a HIV—TAT derived membrane translocation peptide[J].Bioconjug Chem,2000,11(3): 301 - 305.
115 Bullok KE,Dyszlewski M,Prior JL,et al.Characterization of novel histidine- tagged TAT—peptide complexes dual —labeled with 99 mTc— tricarbonyl and fluorescein for scintigraphy and fluorescence microscopy[J].Bioconjug Chem,2002,13(6): 1226-1237.
116 Kaufman CL,Williams M,Ryle LM,et al.Superparamagnetic iron oxide particles transactivator protein- fluorescein isothiocyanate particle labeling for in vivo magnetic resonance imaging detection of cell migration: uptake and durability[J].Transplantation,2003,76(7): 1043- 1046.
117 Lewin M,Carlesso N,Tung CH,et al.TAT peptide - derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells[J].Nat Biotechnol,2000,18(4):410—414.
118 Liu J,Zhang Q,Remsen EE,et al.Nanostructured materials designed for cell binding and transduction[J].Biomacromolecules,2001,2(2):362—368.
119 Dodd CH,Hsu HC,Chu WJ,et al.Normal T-cell response and in vivo magnetic resonance imaging of T cellsloaded with HIV transactivator- peptide—derived superparamagnetic nanoparticlles[J].J Immunol Methods,2001,256(1- 2):89— 105.
120 Tkachenko AG,Xie H,Liu Y,et al.Cellular trajectories of peptide- modified gold particle complexes:comparison of nuclear localization signals and peptide transduction domains[J].Bioconjug Chem,2004,15(3):482—490.
121 Josephson L,Tung CH,Moore A,et al.High—efficiency intracellular magnetic labeling with novel superparamagnetic—TAT peptide conjugates[J].Bioconjug Chem, 1999,10(2): 186—191.
122 TorchiLin VP,Rammohan R,Weissig V,et al.TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low tern perature and in the presence of metabolic inhibitors[J].Proc Natl Acad,2001, 98(15):8786- 8791.
123 Levchenko TS,Rammohan R,Volodina N,et al.TAT peptide — mediated intracellular delivery ofliposomes[J].Methods Enzymol,2003,372:339-349.
124 Marty C,Meylan C,Schott H,et al.Enhanced heparan sulfate proteoglycan— mediated uptake of cell—penetrating peptide—modified liposomes[J]. Cell Mol Life Sci,2004,61(14):1785～1794.
125 Melan MA,Sluder G.Redistribution and differential extraction of soluble proteins in permeabilized cultured cells,Implications for immunofluorescence microscopy[J].J Cell Sci,1992,101(Pt4):731～743.
126 Pichon C,Monsigny M., Roche A.C.. IntracelluLar localization of oligonucleotides:influence of fixative protocols[J].Antisense Nucleic Acid Drug Dev,1999,9(1):89～93.
127 Lundberg M,Johansson M.Positively charged DNA—binding proteins cause apparent cellmembrane translocation[J].Biochem Biophys Res Commun,2002, 291(2):367～371.
128 Lundberg M,Johansson M.Is VP22 nuclear homing an artifact?[J].Nat Biotechnol,2001,19(8):713～714.
129 Richard JP,Melikov K,Vives E,et al.Cell—penetrating peptides: a reevaluation of the mechanism of cellular uptake[J]. J Biol Chem,2003,278(1):585～590.
130 Lundberg M,Wikstrom S,Johansson M.Cell surface adherence and endocytosis of protein transduction domains[J].Mol Ther,2003,8(1): 143～150.
131 Drin G,Cottin S,Blanc E,et al.Studies on the internalisation mechanism of cationic cell—penetrating peptides[J].J Biol Chem,2003,278(33):31192～31201.
132 Ho A,Schwarze SR,Mermelstein SJ,et al.Synthetic protein transduction domains:enhanced transduction potential in vitro and in vivo[J].Cancer Res,2001,61(2):474～477.
133 Richard JP,Melikov K,Vives E,et al.Cell—penetrating peptides:a reevaluation of the mechanism of cellular uptake[J].J Biol Chem,2003,278(1):585～590.
134 Lundberg M,Johansson M.Positively charged DNA—binding proteins cause apparent cell membrane translocation.Biochem[J].Biophys Res Commun, 2002,291 (2):367～371.
135 Lundberg M,Wikstrom S,Johansson M.Cell surface adherence and endocytosis of protein transduction domains[J].Mol Ther,2003, 8(1):143～150.
136 Koppelhus U,Awasthi SK, Zachar V,et al.Cell—dependent differential cellular uptake of PNA,peptides,and PNA -peptide conjugates[J].Antisense Nucleic Acid Drug Dev,2002,12(2):51-63.
137 Violini S,Sharma V,Prior JL,et al.Evidence for a plasma membrane mediated permeability barrier to tat basic domain in well - differentiated epithelial cells:Lack of correlation with heparan sulfate[J].Biochemistry 2002,41 (42): 12652-12661.
138 Kramer SD,Wunderli-Allenspach H.No entry for TAT44 —57 into liposomes and intact MDCK cells:novel approach to study membrane permeation of cell - penetrating peptides[J].Biochim Biophys Acta,2003,1609(2):161 - 169.
139 Falnes PO,Wesche J,Olsnes S.Ability of the Tat basic domain and VP22 to mediate cell binding, but not membrane translocation of the diphtheria toxin A— fragment[J].Biochemistry,2001,40(12):4349-4358.
140 Leifert JA,Harkins S,Whitton JL.Full -length proteins attached to the HIV tat protein transduction domain are neither transduced between cells,nor exhibit enhanced immunogenicity[J].Gene Ther,2002,9(21):1422—1428.
141 Fang B,Xu B,Koch P,et al.Intercellular trafficking of VP22 — GFP fusion proteins is not observed in cultured mammalian cells[J].Gene Ther, 1998,5:1420—1424.
142 Caron NJ,Torrente Y,Camirand G,et al.Intracellular delivery of a Tat —GFP fusion protein into muscle cells[J].Mol Ther,2001,3(3):310—318.
143 Bogoyevitch MA,Kendrick TS,Ng DC,et al.Taking the cell by stealth or storm? Protein transduction domains PTDs as versatile vectors for delivery[J].DNA Cell Biol,2002,21(12):879—894.
144 Hallbrink M,Floren A,Elmquist A,et al.Cargo delivery kinetics of cell — penetrating peptides[J].Biochim Biophys Acta,2001,1515(2): 101 — 109.
145 Mai JC,Shen H,Watkins SC,et al. Efficiency of protein transduction is cell type — dependent and is enhanced by dextran sulfate[J].J Biol Chem,2002,277 (33):30208—30218.
146 Tre'hin R,Cell penetrating peptides:uptake,permeation and metab olism in epithelial models[J].PhD Thesis,2004,5:15314.
147 Violini S,Sharma V,Prior JL,et al.Evidence for a plasma membrane- mediated permeability barrier to tat basic domain in well—differentiated epithelial cells:lack of correlation with heparan sulfate[J].Biochemistry,2002, 41(42):12652～12661.
148 Tre'hin R.Cell penetrating peptides:uptake,permeation and metabolism in epithelial models[J].PhD Thesis,2004,5:15314.
149 Elmquist A,Lindgren M,Bartfai T,et al.VE—cadherinderived cell—penetrat ing peptide,pVEC,with carrier functions[J].Exp Cell Res,2001,269(2):237～244.
150 Elmquist A,Langel U.In vitro uptake and stability study of pVEC and its all—D analog[J].Biol Chem,2003,384(3):387～393.
151 Soomets U,Lindgren M,Gallet X,et al.Deletion analogues of transportan[J]. Biochim Biophys Acta,2000,1467(1): 165～176.
152 Lindgren ME,Hallbrink MM,Elmquist AM,et al.Passageof cell—penetrating peptides across a human epithelial cell layer in vitro[J].Biochem J, 2004,377(1):69～76.
153 Bonavia R,Bajetto A,Barbero s,et al.HIV—1 Tat causes apoptotic death and calcium homeostasis aLterations in rat neurons[J].Biochem Biophys Res Commun,2001, 288(2):301～308.
154 Nath A,Psooy K,Martin C,et al.Identification of a human immunodeficiency virus type 1 Tat epitope that is neuroexcitatory and neurotoxic[J].J Virol, 1996,70(3): 1475～1480.
155 Jia H,Lohr M,Jezequel S,et al.Cysteine—rich and basic domain HIV—1 Tat peptides inhibit angiogenesis and induce endothelial cell apoptosis[J]. Biochem Biophys Res Commun,2001,283(2):469～479.
156 Bolton SJ,Jones DN,Darker JG,et al.Cellular uptake and spread of the cell— permeable peptide penetratin in adult rat brain[J].Eur J Neurosci,2000,12(8): 2847～2855.
157 Garcia—Echeverria C,Jiang L,Ramsey TM,et al.A new antennapedia—derived vector for intracellular delivery of exogenous compounds[J].Bioorg Med Chem Lett,2001,11(11):1363～1366.
158 Shahana S, Kampf C,Roomans GM.Effects of the cationic protein poly—L— arginine on airway epithelial cells in vitro[J].Mediators Inflamm,2002,11(3): 141～148.
159 Santana A,HysLop S,Antunes E,et al.Inflmmatory responses induced by poly—L —arginine in rat Lungs in vivo[J].Agents Actions, 1993,39(3—4):104～l10.
160 Koelle DM,Frank JM,Johnson ML,et al.Recognition of herpes simplex virus type 2 tegument proteins by CD4 T cells infiltrating human genital herpes lesions[J].J Virol,1998,72(9):7476～7483.
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