BDNF-PTD融合蛋白逆转大脑缺血导致神经死亡的药效学和机制研究
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摘要
研究背景
血脑屏障主要由血管内皮细胞和脑组织的星状胶质细胞的足突构成,具有内皮细胞之间紧密连接并且缺乏内吞囊的特点,可限制血液和脑组织之间物质的自由交换,因此大多数药物很难通过血脑屏障进入大脑发挥治疗疾病作用。脑源性神经营养因子(Brain-derived neurotrophic factor, BDNF)为生物体内天然的蛋白分子,在神经系统的生长发育中,对神经元的生存,分化起重要作用；具有增强学习记忆等功能,但同样难以通过血脑屏障进入中枢神经系统治疗神经退行性疾病。目前临床上缺少一种有效的,非侵入性的方法使得BDNF通过血脑屏障而发挥生物学效应,因而限制了BDNF在临床上的应用。所以,以何种给药系统使BDNF进入大脑是发挥BDNF临床治疗作用应用的关键。目前在实验室阶段研究的脑靶向给药系统主要包括：
①脑血管内灌注或脑内直接注射给药。
②病毒载体介导基因后的脑内直接注射给药。
③脂质体包裹药物后系统途径给药。
④脑靶向单克隆抗体偶联药物后系统途径给药。
但这些方法有很多的不足：
①脑血管内灌注或脑内注射给药有极大的不顺应性,且给药后,药物分布在很小的脑部位。
②病毒基因介导给药虽然是很好的入脑载体,但并不适合临床上的急性缺血的脑损伤的治疗,因为脑损伤后在很短的时间内给药才能对神经元细胞有保护和修复作用。而且病毒载体对人的免疫系统,及细胞染色体组有副作用。
③用脂质体包裹药物能增加药物的脂溶性,但脑对脂质体的摄入是绝对的和非特异性的,增加了药物因全身分布而带来的副作用和成本。
④单克隆抗体偶联药物进行静脉给药能够使得BDNF通过血脑屏障到达大脑而发挥生物学效应,但这种方法得到的药物,制备过程繁琐,成本高；脑靶向用单克隆抗体需先进行人源化改造。
如何寻找种有效的,非侵入性的给药系统方法,使得BDNF进入大脑发挥生物学效应?
来源于人免疫缺陷病毒的TAT蛋白能够跨膜导入细胞内部的发现,给我们解决这个问题提供了很好的思路。蛋白转导域(PTD)是指那些小于20个氨基酸、带正电荷,可以穿过大多数细胞膜的穿膜肽的一个富含碱性氨基酸区域,带正电荷的多肽片段与蛋白转导功能相关联。可以将大分子运输入几乎所有的哺乳动物细胞内,无需特殊的环境。其对给合物的大小没有严格的限制,已经在肿瘤,免疫治疗等临床前研究中取得了很大的进展。
本实验室前期通过基因工程方法制备了一种能通过血脑屏障的脑源性神经营养因子-PTD融合蛋白(BDNF-PTD),实验证明其能通过血脑屏障。在此基础上,本文研究了BDNF-PTD在动物脑缺血模型中保护神经元功能的药效学及其作用机制。
目的
研究本实验中心制备的BDNF-PTD在动物脑缺血模型中保护神经元功能的初步药效学功能；通过神经功能缺陷评分对非给药组和给药组进行神经功能缺陷评价；研究本实验中心制备的BDNF-PTD在动物脑缺血模型中保护神经元功能的作用机制。
方法
一、建立SD大鼠脑局部缺血模型
大鼠用10%水合氯醛(3ml／100g)腹腔注射麻醉,仰卧固定在手术台上,在颈部正中切开,剪开前筋膜,钝性分离胸锁乳突肌和胸骨舌骨肌之间的间隙,暴露左侧颈总动脉(CCA)和迷走神经,并分离。往远心段找出颈外动脉(ECA)和颈内动脉(ICA),干净分离ECA、ICA。结扎ECA近分叉端和在距分叉处10mm结扎CCA,并在CCA近分叉处套线待用,用动脉夹夹住ICA。在距分叉处5mm的CCA上用眼科剪45°剪一小斜口,插入栓线,通过分叉处进入ICA,提起在CCA近分叉处准备好的线,打好结但不能太紧,栓线能通过为合适,松开ICA处的动脉夹,继续使栓线顺ICA入颅方向推进,直到推进至分叉处17mm为止。将线栓和CCA固定,剪掉多余的栓线,缝合筋膜和皮肤。
根据Bederson评分法进行神经功能缺陷程度的评分。其原则如下：0分：无神经功能缺损症状,行为正常；1分：前肢屈曲；2分：中度神经功能缺损,抵抗对侧推力下降伴前肢屈曲,无转圈行为；3分：重度神经功能缺损,抵抗对侧推力下降伴前肢屈曲,有转圈行为。1、2、3级为模型成功的标准。
二、脑源性神经营养因子融合蛋白(BDNF-PTD)对大鼠局灶性脑缺血治疗作用的初步药效学研究
通过线栓法制作大鼠大脑中动脉栓塞(MACO)模型。永久性栓塞1个小时后腹腔给每只动物模型注射50ug (50ug/ml)的BDNF-PTD,24小时后断头取脑,大脑切片TTC染色,比较栓塞1小时后给药模型组与未给药模型组的脑梗死面积,观察BDNF-PTD保护大脑缺血神经元死亡的情况。
三、脑源性神经营养因子融合蛋白(BDNF-PTD)对大鼠局灶性脑缺血治疗作用的机制研究
BDNF能提高神经元抵抗缺血的能力,具有促进受损神经元的修复,再生,调节神经结构的重建,促进脑损伤后认知功能恢复等作用。其作用的机制可能与两个主要的信号途径有关：磷脂酰肌醇-3-激酶(PI3K)途径和细胞分裂素(丝裂原)活化蛋白激酶(MAPK/ERK)途径。它们通过激活转录蛋白(如cAMP效应元件结合蛋白)而影响基因的表达来促进神经元的生长和存活。而促分裂原活化蛋白激酶和磷脂酰肌醇-3-激酶途径都与BDNF-TrkB受体信号调节有关。我们研制的BDNF-PTD具有逆转神经元死亡的功能,是否也是通过这些通路来发挥作用,这将在本课题研究中进行验证。
通过线栓法制作大鼠大脑中动脉栓塞(MCAO)模型。永久性栓塞1个小时后腹腔给每只动物模型注射50ug (50ug/ml)的BDNF-PTD,24小时后断头取脑蛋白,通过免疫印迹法(WB),验证BDNF-PTD是否可激活ERK1／2细胞外信号调节激酶和磷脂酰肌醇3激酶(PI3K)。
结果
一、成功建立SD大鼠脑局部缺血模型
大鼠大脑缺血模型制作后,栓塞24小时后,发现给药组和未给药模型组、阴性对照组,在神经功能活动上有不同的表现。根据Bederson评分法对每组模型进行神经功能缺陷程度的评分,给药组大部分处于0到1分。而未给药模型组及阴性对照组有抵抗对侧推力下降伴前肢屈曲,和转圈追尾运动,大部分处于2到3分。
二、脑源性神经营养因子融合蛋白(BDNF-PTD)对大鼠局灶性脑缺血神经保护治疗作用的初步研究
大鼠大脑中动脉栓塞模型在栓塞1小时给药,随后24内观察大鼠偏瘫症状,与未给药模型组相比,给药组大鼠偏瘫症状得到明显的改善。永久性栓塞24小时后,大脑冠状切片TTC染色。梗死面积用Mean±SD表示,BDNF-PTD给药组与未给药模型组、阴性对照组,比较结果有显著性差别(F=26.791,P=0.000<0.01)。发现1小时给药模型组大鼠脑坏死区域面积与未给药模型组相比明显减少75%～80%。
三、脑源性神经营养因子融合蛋白(BDNF-PTD)对大鼠局灶性脑缺血治疗作用的机制研究
研究脑源性神经营养因子融合蛋白(BDNF-PTD)对大鼠局灶性脑缺血治疗作用的机制。在本课题中,实验结果证明,BDNF-PTD激活ERK1／2,使其磷酸化,给药模型组中pERK1/2蛋白表达水平较正常组均有显著性升高(P<0.01)；与未给药模型组中pERK1/2蛋白表达水平比较也均有显著性升高(P<0.05)。
另外,实验结果也证明,BDNF-PTD能上调PI3K,给药模型组中PI3K蛋白表达水平较正常组均有显著性升高(P=0.000<0.01)；与未给药模型组中PI3K蛋白表达水平比较也均有显著性升高(P=0.000<0.01)；而未给药模型组与正常组比较差异没有统计学意义(P>0.05)。
结论
一、在大鼠脑缺血模型中,脑源性神经营养因子融合蛋白(BDNF-PTD)能通过血脑屏障并且能发挥生物学效应。跟未给药模型组对比,显示BDNF-PTD能很好的裸护脑缺血后的神经元。
二、在大鼠脑缺血模型中,证实了脑源性神经营养因子融合蛋白(BDNF-PTD)通过激活ERK细胞外信号调节激酶的磷酸化和上调磷脂酰肌醇3激酶(P3K),从而促进神经元细胞的存活。
BACKGROUND
The blood-brain barrier(BBB) is mainly constituted by vascular endothelial cells and glial cells in brain tissue. Endothelial cells lack of endocytosis capsule, which limit free exchange of materials between the blood and brain tissue. Therefore, many drugs such as BDNF, a natural protein molecules in vivo, are difficult to penetrate through the blood-brain barrier. Currently, there isn't any valid, non-invasive approach which mediates BDNF through BBB to exert biological effects. How to delivery BDNF into brain is a key for its clinical application. The delivery systems for drug targeting are as follows now:
1) Cerebrovascular infusion or intracerebral injection of drug.
2) Viral-mediated gene-therapy.
3) Liposome-encapulated drug delivery system.
4) Monoclonal antibody-coupled drugs for brain targeting.
However, there are many shortcomings for these methods:
1) Cerebrovascular perfusion or local administration of drug in the brain has non-compliance, and the drug could only distribute in the injection area.
2) Viral-mediated gene delivery into the brain is an alternative way, but it is not suit for the treatment of acute ischemic brain injury in clinic because of delayed effects. Moreover, the side effects of viral vector on human immune system and chromosome are uncertain.
3) Liposome can increase the hydrophilicity of drugs. However, the brain uptake of liposomes is absolutely non-specific, which may increase the side effects of drug and cost.
4) Coupled with brain-targeting monoclonal antibody, drugs could penetrate the BBB and reach the brain after intravenous administration.However, this method made the manufacture very cumbersome and expensive. How to find an effective, non-invasive delivery way to mediate drugs, such as BDNF into the brain is imminent.
The protein transduction domain (PTD) discovered in Trans-Activator of Transcription(TAT) protein derived from the human immunodeficiency virus can mediate other proteins into cells by transcytosis. Protein transduction domain (PTD) are positively charged oligopeptides with less than 20 amino acids, and can transport almost all macromolecules into mammalian cells,which provides a good way to penetrate durgs through BBB into the brain.
Our previous study has prepared a brain-derived neurotrophic factor-TAT/PTD fusion protein (BDNF-PTD) [9] through genetic engineering, and confirmed that the fusion protein could through the blood-brain barrier via a systemic administration. In the present study, the pharmacodynamic and the mechanism of neuroprotective effects against ischemic brain neuron death in rats by BDNF-PTD have been elucidated.
OBJECTIVE
To study the pharmacodynamics and neuroprotectiove function of BDNF-PTD in the animal model of cerebral ischemia, and compare the neurological deficit through the neurological deficit scores between non-treatment group and treatment group, and explore the mechanism of neuroprotective effects against brain ischemic in rats by BDNF-PTD.
METHODS
1) established a cerebral ischemia animal model induced by middle cerebral arterial occlusion in Sprague-Dawley rats
Adult male Sprague-Dawley rats (weight,250 to 350 g) were purchased from animal center of Southern Medical University. Focal cerebral ischemia was produced by intraluminal MCAO following the method of Longa et al. After fasting overnight, the animal was lightly anesthetized with Chloral hydrate, The left common carotid artery and the left external carotid artery were exposed, and the occipital artery and superior thyroid artery were electrocoagulated. The left pterygopalatine artery was ligated, the left common carotid artery was clamped, and a 4-0 nylon suture was inserted retrogradely via arteriectomy of the external carotid artery into the internal carotid artery. The tip of the suture was rounded near a flame before insertion. The suture was slowly advanced until resistance was felt. The external carotid artery was ligated, and the common carotid artery clamp was released. The skin incision was sutured.The animal was allowed to recover and was kept warm with a heating lamp. The rat was sedated with halothane and killed by decapitation at 24 hours.Neurological status before the animal was killed was measured as described by Longa et al. Some animals in each treatment group died during the night; these animals were replaced in the study and were not included in the calculation of infarct area.
2) A preliminary study of the therapeutic effect of brain-derived neurotrophic factor fusion protein (BDNF-PTD) on focal cerebral ischemia
Permanent embolism in 1 hours through Suture method in rats produced by middle cerebral artery occlusion (MACO) model. and then intraperitoneal injection to each animal models 50ug (50ug/ml) BDNF-PTD, killed by decapitation at 24 hours, the brain slices staining by TTC, comparing the area of cerebral infarction embolization 1 hour delivery model group and model group,the study showed BDNF-PTD can protect ischemic neurons in the brain.
3) The Mechanism Of Reversal the neuron death on brain ischemia by BDNF-PTD
BDNF can enhance the capacity of neurons to resist ischemia and promote repair of damaged neurons, regeneration, regulate neural structure, reconstruction, the promotion of cognition functional recovery after brain injury. The mechanism of its role may be related to the two main signaling pathways, these two signaling pathways has been proven to promote the growth and survival of neurons. Phosphatidylinositol-3-kinase (PI3K) pathways and cytokinins (mitogen)-activated protein kinase (MAPK) signaling pathways, which through activation transcription proteins (such as the cAMP response element binding protein) affect gene expression to promote nerve growth and survival. The mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathway with the BDNF-TrkB receptor signaling. BDNF-PTD has a reverse function of neuronal death, whether it is through these pathways play a role, which will verify in this paper.
Permanent embolism after 1 hours through Suture method in rats produced by middle cerebral artery occlusion (MACO) model. and then intraperitoneal injection to each animal models 50ug (50ug/ml) BDNF-PTD, killed by decapitation at 24 hours, the brain were dissected, complemented with lysis buffer, homogenized, and centrifuged, and supernatants were used for Western blot (WB), verify whether the BDNF-PTD can activate ERK extracellular signal-regulated kinase and phosphatidylinositol 3-kinase (PI3K).
RESULTS
1) The establishment of SD rat model of cerebral ischemia
Cerebral ischemia model is made by middle cerebral artery occlusion (MCAO), the activities in the neurological function have different performance in the durg group and the blank model group, negative control group when 24 hours after vascular occlusion. range of deficit score 0 to 1 in the treatment group. The model group and negative control group, decreased resistance to thrust and with the contralateral forelimb flexion, and turning in circles rear-end movements(range of deficit score 2 to 3).
2) The preliminary study of therapeutic effect of brain-derived neurotrophic factor fusion protein (BDNF-PTD) on focal cerebral ischemia
The hemiplegic symptoms are significantly improved in thrombosis 1 hour group by middle cerebral artery occlusion after administration BDNF-PTD, rather than non-treatment group. TTC stained brain slices and found rat brain necrotic area of 1 hour thrombosi group has significantly reduced compared with the control group. Infarct size Values given are mean±SD, treatment group compare to no treatment group with negative control group, the results statistically significant difference (F= 26.791, P= 0.000<0.01). The necrotic area of treatment model group are significantly reduced(75%～80%) compared with nontreatment model group.
Results show that BDNF-PTD can through the blood-brain barrier and can perform its biological effects compared with the control group, this result found that BDNF-PTD can be well protected neurons survive after cerebral ischemia. 3) The mechanism of therapeutic effect brain-derived neurotrophic factor fusion protein (BDNF-PTD) on focal cerebral ischemia
Studies the mechanism of therapeutic effect of brain-derived neurotrophic factor fusion protein (BDNF-PTD) on focal cerebral ischemia. Focal cerebral ischemia model in rats produced by middle cerebral artery occlusion (MACO). intraperitoneal injection to each animal models 50ug (50ug/ml) BDNF-PTD after Permanent embolism in 1 hours, after 24 hours the animal decapitated and the brain protein analysis by Western blot, results show BDNF-PTD activated ERK through phosphorylation, pERKprotein levels were significantly increased (P<0.01) compared with normal group; and nontreatment model group, pERK protein levels also were significantly elevated (P<0.05).
In addition, results also shows, BDNF-PTD could were significantly upregulate PI3K (P=0.000<0.01) compared with normal group;PI3K levels also also were significantly elevated (P=0.000<0.01)compared with nontreatment model group.but the PI3K levels were not statistically significant (P>0.05) in nontreatment model group between normal group.
CONCLUSION
1) The embolism hemiplegia symptoms are significantly improved in middle cerebral artery occlusion model after treat by BDNF-PTD. Study found that brain necrotic area were significantly reduced in treatment group compared with the control group. As compared with the model group, BDNF-PTD can pass through the blood-brain barrier and perform its biological effects which demonstrate that BDNF-PTD can be well protected neurons in cerebral ischemia.
2) To study the mechanism of therapeutic effect of brain-derived neurotrophic factor fusion protein (BDNF-PTD) on focal cerebral ischemia. produced middle cerebral artery occlusion (MACO) model through Suture method in rats. after 24 hours the rats decapitated and brain protein analysis by Western blot, the results confirmed that the BDNF-PTD can activated ERK extracellular signal-regulated kinase and upregulate phosphatidylinositol 3-kinase (PI-3K).
血脑屏障主要由血管内皮细胞和脑组织的星状胶质细胞的足突构成,具有内皮细胞之间紧密连接并且缺乏内吞囊的特点,可限制血液和脑组织之间物质的自由交换,因此大多数药物很难通过血脑屏障进入大脑发挥治疗疾病作用。脑源性神经营养因子(Brain-derived neurotrophic factor, BDNF)为生物体内天然的蛋白分子,在神经系统的生长发育中,对神经元的生存,分化起重要作用；具有增强学习记忆等功能,但同样难以通过血脑屏障进入中枢神经系统治疗神经退行性疾病。目前临床上缺少一种有效的,非侵入性的方法使得BDNF通过血脑屏障而发挥生物学效应,因而限制了BDNF在临床上的应用。所以,以何种给药系统使BDNF进入大脑是发挥BDNF临床治疗作用应用的关键。目前在实验室阶段研究的脑靶向给药系统主要包括：
①脑血管内灌注或脑内直接注射给药。
②病毒载体介导基因后的脑内直接注射给药。
③脂质体包裹药物后系统途径给药。
④脑靶向单克隆抗体偶联药物后系统途径给药。
但这些方法有很多的不足：
①脑血管内灌注或脑内注射给药有极大的不顺应性,且给药后,药物分布在很小的脑部位。
②病毒基因介导给药虽然是很好的入脑载体,但并不适合临床上的急性缺血的脑损伤的治疗,因为脑损伤后在很短的时间内给药才能对神经元细胞有保护和修复作用。而且病毒载体对人的免疫系统,及细胞染色体组有副作用。
③用脂质体包裹药物能增加药物的脂溶性,但脑对脂质体的摄入是绝对的和非特异性的,增加了药物因全身分布而带来的副作用和成本。
④单克隆抗体偶联药物进行静脉给药能够使得BDNF通过血脑屏障到达大脑而发挥生物学效应,但这种方法得到的药物,制备过程繁琐,成本高；脑靶向用单克隆抗体需先进行人源化改造。
如何寻找种有效的,非侵入性的给药系统方法,使得BDNF进入大脑发挥生物学效应?
来源于人免疫缺陷病毒的TAT蛋白能够跨膜导入细胞内部的发现,给我们解决这个问题提供了很好的思路。蛋白转导域(PTD)是指那些小于20个氨基酸、带正电荷,可以穿过大多数细胞膜的穿膜肽的一个富含碱性氨基酸区域,带正电荷的多肽片段与蛋白转导功能相关联。可以将大分子运输入几乎所有的哺乳动物细胞内,无需特殊的环境。其对给合物的大小没有严格的限制,已经在肿瘤,免疫治疗等临床前研究中取得了很大的进展。
本实验室前期通过基因工程方法制备了一种能通过血脑屏障的脑源性神经营养因子-PTD融合蛋白(BDNF-PTD),实验证明其能通过血脑屏障。在此基础上,本文研究了BDNF-PTD在动物脑缺血模型中保护神经元功能的药效学及其作用机制。
目的
研究本实验中心制备的BDNF-PTD在动物脑缺血模型中保护神经元功能的初步药效学功能；通过神经功能缺陷评分对非给药组和给药组进行神经功能缺陷评价；研究本实验中心制备的BDNF-PTD在动物脑缺血模型中保护神经元功能的作用机制。
方法
一、建立SD大鼠脑局部缺血模型
大鼠用10%水合氯醛(3ml／100g)腹腔注射麻醉,仰卧固定在手术台上,在颈部正中切开,剪开前筋膜,钝性分离胸锁乳突肌和胸骨舌骨肌之间的间隙,暴露左侧颈总动脉(CCA)和迷走神经,并分离。往远心段找出颈外动脉(ECA)和颈内动脉(ICA),干净分离ECA、ICA。结扎ECA近分叉端和在距分叉处10mm结扎CCA,并在CCA近分叉处套线待用,用动脉夹夹住ICA。在距分叉处5mm的CCA上用眼科剪45°剪一小斜口,插入栓线,通过分叉处进入ICA,提起在CCA近分叉处准备好的线,打好结但不能太紧,栓线能通过为合适,松开ICA处的动脉夹,继续使栓线顺ICA入颅方向推进,直到推进至分叉处17mm为止。将线栓和CCA固定,剪掉多余的栓线,缝合筋膜和皮肤。
根据Bederson评分法进行神经功能缺陷程度的评分。其原则如下：0分：无神经功能缺损症状,行为正常；1分：前肢屈曲；2分：中度神经功能缺损,抵抗对侧推力下降伴前肢屈曲,无转圈行为；3分：重度神经功能缺损,抵抗对侧推力下降伴前肢屈曲,有转圈行为。1、2、3级为模型成功的标准。
二、脑源性神经营养因子融合蛋白(BDNF-PTD)对大鼠局灶性脑缺血治疗作用的初步药效学研究
通过线栓法制作大鼠大脑中动脉栓塞(MACO)模型。永久性栓塞1个小时后腹腔给每只动物模型注射50ug (50ug/ml)的BDNF-PTD,24小时后断头取脑,大脑切片TTC染色,比较栓塞1小时后给药模型组与未给药模型组的脑梗死面积,观察BDNF-PTD保护大脑缺血神经元死亡的情况。
三、脑源性神经营养因子融合蛋白(BDNF-PTD)对大鼠局灶性脑缺血治疗作用的机制研究
BDNF能提高神经元抵抗缺血的能力,具有促进受损神经元的修复,再生,调节神经结构的重建,促进脑损伤后认知功能恢复等作用。其作用的机制可能与两个主要的信号途径有关：磷脂酰肌醇-3-激酶(PI3K)途径和细胞分裂素(丝裂原)活化蛋白激酶(MAPK/ERK)途径。它们通过激活转录蛋白(如cAMP效应元件结合蛋白)而影响基因的表达来促进神经元的生长和存活。而促分裂原活化蛋白激酶和磷脂酰肌醇-3-激酶途径都与BDNF-TrkB受体信号调节有关。我们研制的BDNF-PTD具有逆转神经元死亡的功能,是否也是通过这些通路来发挥作用,这将在本课题研究中进行验证。
通过线栓法制作大鼠大脑中动脉栓塞(MCAO)模型。永久性栓塞1个小时后腹腔给每只动物模型注射50ug (50ug/ml)的BDNF-PTD,24小时后断头取脑蛋白,通过免疫印迹法(WB),验证BDNF-PTD是否可激活ERK1／2细胞外信号调节激酶和磷脂酰肌醇3激酶(PI3K)。
结果
一、成功建立SD大鼠脑局部缺血模型
大鼠大脑缺血模型制作后,栓塞24小时后,发现给药组和未给药模型组、阴性对照组,在神经功能活动上有不同的表现。根据Bederson评分法对每组模型进行神经功能缺陷程度的评分,给药组大部分处于0到1分。而未给药模型组及阴性对照组有抵抗对侧推力下降伴前肢屈曲,和转圈追尾运动,大部分处于2到3分。
二、脑源性神经营养因子融合蛋白(BDNF-PTD)对大鼠局灶性脑缺血神经保护治疗作用的初步研究
大鼠大脑中动脉栓塞模型在栓塞1小时给药,随后24内观察大鼠偏瘫症状,与未给药模型组相比,给药组大鼠偏瘫症状得到明显的改善。永久性栓塞24小时后,大脑冠状切片TTC染色。梗死面积用Mean±SD表示,BDNF-PTD给药组与未给药模型组、阴性对照组,比较结果有显著性差别(F=26.791,P=0.000<0.01)。发现1小时给药模型组大鼠脑坏死区域面积与未给药模型组相比明显减少75%～80%。
三、脑源性神经营养因子融合蛋白(BDNF-PTD)对大鼠局灶性脑缺血治疗作用的机制研究
研究脑源性神经营养因子融合蛋白(BDNF-PTD)对大鼠局灶性脑缺血治疗作用的机制。在本课题中,实验结果证明,BDNF-PTD激活ERK1／2,使其磷酸化,给药模型组中pERK1/2蛋白表达水平较正常组均有显著性升高(P<0.01)；与未给药模型组中pERK1/2蛋白表达水平比较也均有显著性升高(P<0.05)。
另外,实验结果也证明,BDNF-PTD能上调PI3K,给药模型组中PI3K蛋白表达水平较正常组均有显著性升高(P=0.000<0.01)；与未给药模型组中PI3K蛋白表达水平比较也均有显著性升高(P=0.000<0.01)；而未给药模型组与正常组比较差异没有统计学意义(P>0.05)。
结论
一、在大鼠脑缺血模型中,脑源性神经营养因子融合蛋白(BDNF-PTD)能通过血脑屏障并且能发挥生物学效应。跟未给药模型组对比,显示BDNF-PTD能很好的裸护脑缺血后的神经元。
二、在大鼠脑缺血模型中,证实了脑源性神经营养因子融合蛋白(BDNF-PTD)通过激活ERK细胞外信号调节激酶的磷酸化和上调磷脂酰肌醇3激酶(P3K),从而促进神经元细胞的存活。
BACKGROUND
The blood-brain barrier(BBB) is mainly constituted by vascular endothelial cells and glial cells in brain tissue. Endothelial cells lack of endocytosis capsule, which limit free exchange of materials between the blood and brain tissue. Therefore, many drugs such as BDNF, a natural protein molecules in vivo, are difficult to penetrate through the blood-brain barrier. Currently, there isn't any valid, non-invasive approach which mediates BDNF through BBB to exert biological effects. How to delivery BDNF into brain is a key for its clinical application. The delivery systems for drug targeting are as follows now:
1) Cerebrovascular infusion or intracerebral injection of drug.
2) Viral-mediated gene-therapy.
3) Liposome-encapulated drug delivery system.
4) Monoclonal antibody-coupled drugs for brain targeting.
However, there are many shortcomings for these methods:
1) Cerebrovascular perfusion or local administration of drug in the brain has non-compliance, and the drug could only distribute in the injection area.
2) Viral-mediated gene delivery into the brain is an alternative way, but it is not suit for the treatment of acute ischemic brain injury in clinic because of delayed effects. Moreover, the side effects of viral vector on human immune system and chromosome are uncertain.
3) Liposome can increase the hydrophilicity of drugs. However, the brain uptake of liposomes is absolutely non-specific, which may increase the side effects of drug and cost.
4) Coupled with brain-targeting monoclonal antibody, drugs could penetrate the BBB and reach the brain after intravenous administration.However, this method made the manufacture very cumbersome and expensive. How to find an effective, non-invasive delivery way to mediate drugs, such as BDNF into the brain is imminent.
The protein transduction domain (PTD) discovered in Trans-Activator of Transcription(TAT) protein derived from the human immunodeficiency virus can mediate other proteins into cells by transcytosis. Protein transduction domain (PTD) are positively charged oligopeptides with less than 20 amino acids, and can transport almost all macromolecules into mammalian cells,which provides a good way to penetrate durgs through BBB into the brain.
Our previous study has prepared a brain-derived neurotrophic factor-TAT/PTD fusion protein (BDNF-PTD) [9] through genetic engineering, and confirmed that the fusion protein could through the blood-brain barrier via a systemic administration. In the present study, the pharmacodynamic and the mechanism of neuroprotective effects against ischemic brain neuron death in rats by BDNF-PTD have been elucidated.
OBJECTIVE
To study the pharmacodynamics and neuroprotectiove function of BDNF-PTD in the animal model of cerebral ischemia, and compare the neurological deficit through the neurological deficit scores between non-treatment group and treatment group, and explore the mechanism of neuroprotective effects against brain ischemic in rats by BDNF-PTD.
METHODS
1) established a cerebral ischemia animal model induced by middle cerebral arterial occlusion in Sprague-Dawley rats
Adult male Sprague-Dawley rats (weight,250 to 350 g) were purchased from animal center of Southern Medical University. Focal cerebral ischemia was produced by intraluminal MCAO following the method of Longa et al. After fasting overnight, the animal was lightly anesthetized with Chloral hydrate, The left common carotid artery and the left external carotid artery were exposed, and the occipital artery and superior thyroid artery were electrocoagulated. The left pterygopalatine artery was ligated, the left common carotid artery was clamped, and a 4-0 nylon suture was inserted retrogradely via arteriectomy of the external carotid artery into the internal carotid artery. The tip of the suture was rounded near a flame before insertion. The suture was slowly advanced until resistance was felt. The external carotid artery was ligated, and the common carotid artery clamp was released. The skin incision was sutured.The animal was allowed to recover and was kept warm with a heating lamp. The rat was sedated with halothane and killed by decapitation at 24 hours.Neurological status before the animal was killed was measured as described by Longa et al. Some animals in each treatment group died during the night; these animals were replaced in the study and were not included in the calculation of infarct area.
2) A preliminary study of the therapeutic effect of brain-derived neurotrophic factor fusion protein (BDNF-PTD) on focal cerebral ischemia
Permanent embolism in 1 hours through Suture method in rats produced by middle cerebral artery occlusion (MACO) model. and then intraperitoneal injection to each animal models 50ug (50ug/ml) BDNF-PTD, killed by decapitation at 24 hours, the brain slices staining by TTC, comparing the area of cerebral infarction embolization 1 hour delivery model group and model group,the study showed BDNF-PTD can protect ischemic neurons in the brain.
3) The Mechanism Of Reversal the neuron death on brain ischemia by BDNF-PTD
BDNF can enhance the capacity of neurons to resist ischemia and promote repair of damaged neurons, regeneration, regulate neural structure, reconstruction, the promotion of cognition functional recovery after brain injury. The mechanism of its role may be related to the two main signaling pathways, these two signaling pathways has been proven to promote the growth and survival of neurons. Phosphatidylinositol-3-kinase (PI3K) pathways and cytokinins (mitogen)-activated protein kinase (MAPK) signaling pathways, which through activation transcription proteins (such as the cAMP response element binding protein) affect gene expression to promote nerve growth and survival. The mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathway with the BDNF-TrkB receptor signaling. BDNF-PTD has a reverse function of neuronal death, whether it is through these pathways play a role, which will verify in this paper.
Permanent embolism after 1 hours through Suture method in rats produced by middle cerebral artery occlusion (MACO) model. and then intraperitoneal injection to each animal models 50ug (50ug/ml) BDNF-PTD, killed by decapitation at 24 hours, the brain were dissected, complemented with lysis buffer, homogenized, and centrifuged, and supernatants were used for Western blot (WB), verify whether the BDNF-PTD can activate ERK extracellular signal-regulated kinase and phosphatidylinositol 3-kinase (PI3K).
RESULTS
1) The establishment of SD rat model of cerebral ischemia
Cerebral ischemia model is made by middle cerebral artery occlusion (MCAO), the activities in the neurological function have different performance in the durg group and the blank model group, negative control group when 24 hours after vascular occlusion. range of deficit score 0 to 1 in the treatment group. The model group and negative control group, decreased resistance to thrust and with the contralateral forelimb flexion, and turning in circles rear-end movements(range of deficit score 2 to 3).
2) The preliminary study of therapeutic effect of brain-derived neurotrophic factor fusion protein (BDNF-PTD) on focal cerebral ischemia
The hemiplegic symptoms are significantly improved in thrombosis 1 hour group by middle cerebral artery occlusion after administration BDNF-PTD, rather than non-treatment group. TTC stained brain slices and found rat brain necrotic area of 1 hour thrombosi group has significantly reduced compared with the control group. Infarct size Values given are mean±SD, treatment group compare to no treatment group with negative control group, the results statistically significant difference (F= 26.791, P= 0.000<0.01). The necrotic area of treatment model group are significantly reduced(75%～80%) compared with nontreatment model group.
Results show that BDNF-PTD can through the blood-brain barrier and can perform its biological effects compared with the control group, this result found that BDNF-PTD can be well protected neurons survive after cerebral ischemia. 3) The mechanism of therapeutic effect brain-derived neurotrophic factor fusion protein (BDNF-PTD) on focal cerebral ischemia
Studies the mechanism of therapeutic effect of brain-derived neurotrophic factor fusion protein (BDNF-PTD) on focal cerebral ischemia. Focal cerebral ischemia model in rats produced by middle cerebral artery occlusion (MACO). intraperitoneal injection to each animal models 50ug (50ug/ml) BDNF-PTD after Permanent embolism in 1 hours, after 24 hours the animal decapitated and the brain protein analysis by Western blot, results show BDNF-PTD activated ERK through phosphorylation, pERKprotein levels were significantly increased (P<0.01) compared with normal group; and nontreatment model group, pERK protein levels also were significantly elevated (P<0.05).
In addition, results also shows, BDNF-PTD could were significantly upregulate PI3K (P=0.000<0.01) compared with normal group;PI3K levels also also were significantly elevated (P=0.000<0.01)compared with nontreatment model group.but the PI3K levels were not statistically significant (P>0.05) in nontreatment model group between normal group.
CONCLUSION
1) The embolism hemiplegia symptoms are significantly improved in middle cerebral artery occlusion model after treat by BDNF-PTD. Study found that brain necrotic area were significantly reduced in treatment group compared with the control group. As compared with the model group, BDNF-PTD can pass through the blood-brain barrier and perform its biological effects which demonstrate that BDNF-PTD can be well protected neurons in cerebral ischemia.
2) To study the mechanism of therapeutic effect of brain-derived neurotrophic factor fusion protein (BDNF-PTD) on focal cerebral ischemia. produced middle cerebral artery occlusion (MACO) model through Suture method in rats. after 24 hours the rats decapitated and brain protein analysis by Western blot, the results confirmed that the BDNF-PTD can activated ERK extracellular signal-regulated kinase and upregulate phosphatidylinositol 3-kinase (PI-3K).
引文
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