重构型自发活化Caspase3基因的构建及其在骨癌痛大鼠的镇痛效应研究
摘要
研究背景
癌痛的治疗一直是临床上的难题,随着对恶性肿瘤的诊断和治疗技术的不断进步,癌性疼痛已经成为严重影响患者生活质量和生存率的主要问题。75%-90%的转移性恶性肿瘤患者会发生严重的癌性疼痛。恶性肿瘤,如前列腺癌,乳腺癌和肺癌等,均易发生骨转移,引起病理性骨痛,即骨癌痛。骨癌痛性质严重,而且发生率极高。目前对于骨癌痛的治疗主要依赖阿片类药物,但随着时间的延长,其用量逐渐增大,并产生严重的副作用,如成瘾,耐受,恶心,呕吐,便秘和呼吸抑制等。因此极有必要寻求一种基于疼痛机制性的,具有高效、作用持久、毒副作用小等优点的新的治疗方案。
骨癌痛动物模型的建立为研究者提供了研究骨癌痛复杂机制和探索更有效靶向药物治疗的契机,特别是大鼠胫骨转移性癌痛模型,其与人类骨癌痛的发生机制有诸多相似之处,能较好的模拟人类转移性骨癌痛的发生,具有重大的研究意义。在此实验模型中,侵袭性肿瘤细胞被注射到大鼠股骨或胫骨骨髓腔。随着肿瘤的生长和骨质破坏,可产生一系列行为学,细胞学和神经生化学改变。Walker256乳腺癌细胞具有明显的骨侵袭能力,常用于建立大鼠转移性胫骨癌痛模型。因此,本研究拟采用Walker256乳腺癌细胞建立大鼠胫骨转移癌痛模型,为下一步的研究奠定基础。
脑干中的某些结构会向脊髓发出“下行，，信号,调控来自脊髓背角的痛觉信息向中枢的传递,并参于各种痛觉相关信息的整合和处理,是疼痛调节的高级中枢。因此,在脊髓上水平完全阻断伤害性信号的上行或下行性传导,有望成为治疗顽固性疼痛的理想策略。延髓头端腹内侧区(rostral ventromedial medulla, RVM)是脑干下行调控系统的重要结构,其内有富含5—羟色胺的中缝大核(nucleus raphe magnus, NRM),网状巨细胞核α部(gigantocellularis pars alpha)和外侧网状巨细胞核等神经核团。RVM区接受并整合来自中脑导水管周围灰质(periaqueductal gray, PAG)的信息,其内神经元轴突沿脊髓背侧束(dorsolateral funiculus, DLF)下行并终于脊髓,构成了疼痛信号下行调控系统的主要输出通路,并能易化或抑制脊髓痛觉信号传递。因此,毁损RVM区易化神经元是削弱慢性疼痛下行易化调控系统作用,治疗顽固性疼痛的有效方法。
以往有研究者利用颅内微注射的方法,在慢性疼痛模型大鼠RVM区内注射利多卡因封闭其易化神经元的功能,虽然达到了良好的镇痛效果,但随着药物代谢,其镇痛作用逐渐消失；也有研究者将神经毒素—皂角素注射到慢性疼痛模型大鼠的RVM区,毁损RVM区神经元,这种方法虽然可以获得持久的镇痛效果,但由于神经元坏死,不可避免的会诱发周围神经组织炎性免疫反应。RVM区调控机体呼吸,心率,血压,体温,运动等重要生理功能。神经元坏死产生的化学物质所诱发的炎性及免疫反应,很可能对机体正常生理功能产生严重的影响。
Caspase 3是多数细胞凋亡途径中的末端效应蛋白酶,但其活化需要外源刺激及上游一系列caspase分子级联反应。Srinivasula等通过分子生物学的方法模拟caspase3在细胞内的活化过程,将其大、小亚基序列颠倒后,构建了无需外源刺激或上游caspase分子作用,就可自发活化的人源性重组caspase3基因,直接介导靶细胞凋亡,可作为基因治疗领域一个极好的细胞致死基因。但人源性基因可能会诱发实验大鼠免疫反应,干扰实验结果的准确性,因此,本研究拟通过构建大鼠源性重组Caspase3基因,并将其与非病毒载体PEI共同定向微注射到胫骨转移癌痛模型大鼠RVM区,启动RVM区神经元自发凋亡,达到“永久”性治疗骨癌痛的效果。
研究方法与结果
1、大鼠源性重组caspase3基因的构建及促凋亡效应评价
方法：提取大鼠星形胶质瘤C6细胞总RNA后,通过逆转录PCR的方法得到cDNA。根据Genbank中caspase3基因的序列分别设计其大、小亚基的引物,并通过重组PCR的方法获得大小亚基顺序颠倒的大鼠源性重组caspase3基因(Remcasp3)。将Remcasp3分别定向克隆到真核表达载体pcDNA3.1+和C1后分别转染人胚肾293T细胞和大鼠永生化神经干细胞,观察细胞形态学变化,通过Annexin V-PI双染、流式细胞术、MTT法来检测其促细胞凋亡作用,透射电镜检测其凋亡微形态学改变。
结果：成功构建出大小亚基顺序颠倒的大鼠源性重组caspase3基因片段,测序结果与预期结果相一致。转染C1-Remcasp3后,INPC及293T出现明显凋亡细胞。电镜下可见胞质浓缩,核染色质边集,并有凋亡小体的形成等典型的细胞凋亡的特征。MTT检测结果提示两种细胞的增殖明显被抑制(INPC:44.61±0.15%; 293T:48.35±0.16%),与对照组相比有统计学差异(P<0.05)。Annexin V-PI共染色流式细胞术检测两种细胞的凋亡率(INPC:16.002±0.99%; 293T:30.667±1.52%),与对照组相比有统计学差异(P<0.05)。
2、大鼠骨癌痛模型的构建及评价
方法：将培养的Walker 256大鼠乳腺癌细胞注入大鼠腹腔内。接种6-7d后收集癌性腹水,经处理后用于胫骨接种。雌性SD大鼠32只,随机分为4组,每组8只。Ⅰ为正常对照组；Ⅱ为D-Hank's组：将10μl无菌D-Hank's溶液注射至大鼠右侧胫骨上段骨髓腔；Ⅲ为热灭活细胞组：于大鼠右侧胫骨上段骨髓腔注射热灭活的Walker256乳腺癌细胞悬液10μl；Ⅳ为骨癌痛模型组：于大鼠右侧胫骨上段骨髓腔注射有活性的Walker256乳腺癌细胞悬液10μl。分别于模型制备前一天,当天,至术后第十五天,每天检测机械性痛觉超敏,机械性痛觉过敏和移动诱发痛,并于模型制备后第15天进行后肢X射线检查。放射学检查后,分别进行大鼠脊髓GFAP,OX42免疫荧光染色和脑干RVM区及脊髓匀浆高效液相色谱(high performance liquid chromatography, HPLC)检测中枢神经系统谷氨酸的浓度。
结果：骨癌痛模型组大鼠出现了明显的痛觉行为学改变,与对照组相比差异有统计学意义(P＜0.05)。接种了有活性的肿瘤细胞后第十五天进行放射学检查,在术侧胫骨注射部位及周围出现较严重的骨质破坏,骨皮质缺失,而对照组大鼠术侧骨皮质完好,未发现骨质破坏。激光共聚焦检测大鼠脊髓GFAP,OX42免疫荧光发现,骨癌痛组大鼠GFAP标记的星形胶质细胞和OX42标记的小胶质细胞明显活化,胞体增大,数量增多。高效液相色谱结果显示：骨癌痛组大鼠RVM区和脊髓兴奋性氨基酸谷氨酸含量均显著升高,与对照组相比差异有统计学意义(P＜0.05)。
3、RVM区靶向注射In-vivo jetPEI/重组caspase3基因复合物在骨癌痛大鼠的镇痛效应评价
方法：鉴定pcDNA3.1+-Remcasp3单克隆菌种,扩增后进行性质粒大量制备,并计算质粒DNA的纯度和浓度。在无菌条件下将质粒DNA与非病毒载体In-vivo jetPEI按一定比例混匀后溶于10%无菌葡萄糖溶液中,再加入无菌双蒸水使葡萄糖的终浓度为5%。在实验大鼠接种有活性的肿瘤细胞10～12天后,以丙泊酚麻醉实验大鼠(50μg/100g i.p.),置于脑立体定位仪上,依据RVM区的解剖位置(前后位：距前囟11mm；左右位：距正中线各旁开0.6mm；背腹位：距颅骨8.5mm)。将药物缓慢注射入双侧RVM区(左右各1μl)。停留片刻后,缓慢退出微量加样器。分别在立体定位注射前一天,当天,至手术后第七天,每天进行痛觉行为学检测。立体定位注射DNA/PEI混合物后48h,分别取Remcasp3组和正常对照组大鼠RVM区,制备组织块石蜡切片,进行Tunel检测；制备脊髓切片进行GFAP,OX42免疫荧光染色；取脑干RVM区及脊髓匀浆以高效液相色谱(HPLC)法检测中枢神经系统谷氨酸的浓度。
结果：RVM区内微注射前,各组骨癌痛大鼠机械缩爪阈值,缩爪反应时间和移动诱发痛觉评分无统计学差异。RVM区内立体定位注射重组质粒／PEI混合物24h后,大鼠机械性缩爪阈值明显升高,缩爪反应时间明显缩短,移动诱发痛觉评分显著降低,差异有统计学意义(P<0.05)。Tunel检测结果显示RVM区可见大量凋亡细胞。HPLC检测结果显示骨癌痛组大鼠RVM区和脊髓内谷氨酸含量在靶向注射重组自杀基因／PEI复合物后显著下降。与癌痛组相比,差异有统计学意义(P＜0.05)。在免疫荧光实验中,骨癌痛组大鼠GFAP标记的星形胶质细胞和OX42标记的小胶质细胞明显活化,数量增多,而颅内微注射重组质粒／PEI复合物后,星形胶质细胞和小胶质细胞的活化明显受到抑制,活化细胞数量显著减少。
4、RVM区内靶向注射重组caspase3/In-vivo jetPEI的安全性评价
方法：分别于RVM区微注射前,及手术后当天,第1天,第2天,第3天至第7天分别测量Naive组,PEI组,5%Glucose组,PEI+5%Glucose组和Remcasp3组大鼠体重,直肠温度,呼吸频率和心率值。
结果：术前各组大鼠生理参数基础值无统计学差异,RVM区微注射后整个观察期内,各组大鼠体重、直肠温度、呼吸频率、心率等生命体征参数之间均无显著差异(P＞0.05)。
5、统计学处理
采用SigmaStat 3.0统计软件进行处理。计量资料以均数±标准差((?)±s)表示,行为学测定及其余计量资料采用单因素方差分析。P＜0.05为差异有统计学意义。
研究总结
一主要研究结果
1、大鼠源性重组caspase3基因可在真核细胞中表达,并在无外源性刺激下直接诱导细胞发生凋亡。
2、RVM区内注射In-vivo jetPEI/重组caspase3基因复合物能诱发其神经元凋亡,并可对骨癌痛大鼠产生显著,完善,持久的镇痛效果。
3、骨癌痛大鼠中枢神经系统兴奋性氨基酸谷氨酸含量显著升高,脊髓星形胶质细胞,小胶质细胞明显活化,数量增多；诱导RVM区神经元凋亡后,中枢神经系统谷氨酸水平显著下降,脊髓星形胶质细胞,小胶质细胞活化水平显著下降。
4、RVM区单次注射,并且诱导其内神经元凋亡不会对实验大鼠的运动功能和生理功能产生显著的影响。
二、研究结论
1、本研究构建的大鼠源性重组caspase3基因能够在真核细胞中表达,并在无外源刺激和上游活化的情况下直接诱导神经细胞与非神经细胞自发凋亡,为基于免疫功能正常的实验大鼠相关疾病的研究和治疗提供一种新型有效的细胞杀伤基因,并为神经系统难治性疾病的基因治疗提供了又一种有效的目的基因。
2、与神经病理性痛和炎性痛的发生机制相似,RVM区在骨癌痛的产生和维持中也起到重要作用。以凋亡的方式毁损RVM区易化神经元能够起到有效并且安全的镇痛作用。
3、非病毒阳离子载体PEI能将目的基因安全,高效地转染至中枢神经系统。
4、中枢神经系统兴奋性氨基酸谷氨酸水平,脊髓星形胶质细胞,小胶质细胞的活化状态与骨癌痛的发生发展显著相关。
5、立体定位引导下RVM区单次注射DNA/PEI混合物未对实验动物的生理功能产生明显影响,提示RVM核团的靶向毁损可以发挥有效、持久和完善的镇痛效果,且具有较高的安全性,为顽固性疼痛治疗提供了一个有效的脊髓上治疗靶点。
Background
Cancer-related pain has always been a difficult clinical issue. As great improvements have been made in the detection and treatment of most types of cancer, cancer pain have played a significant negative role in the quality of lives and the survival rates of cancer patients. Between 75 and 90% of patients with metastatic cancer will experience significant cancer-induced pain. Cancers, including prostate cancer, breast cancer, and lung cancer, have special affinities to metastasize to bone, and produce pathological bone cancer pain. Bone cancer pain is one of the most severe cancer induced pain with high morbidity. The treatment of bone cancer pain now has been largely based on the use of opioids. Opioids are effective in attenuating bone cancer induced pain, but with the increases in frequence and doses, the analgesic effects from opioids are frequently accompanied by side effects such as addiction, tolerance, nausea, vomiting, constipation and respiratory depression. Therefore, it is very necessary to explore a novel mechanism-based treatment which has more effective, long-lasting and less side-effects to relieve the bone cancer pain.
The recently published animal models of bone cancer pain have allowed the researchers greater insights into the complicated mechanisms of bone cancer pain and the explore more effective targeted drug managements. The metastatic rat tibia bone cancer pain presents the similar characteristic pain symptoms to human bone cancer pain. In this model, metastative cancer cells were injected into the intramedullary cavity of femur or tibia of rats to produce a serie of behavioral, cellular, and neurochemical changes correlated with the development of cancer growth and bone destruction. Walker 256 carcinoma cells have significant bone metastatic affinities, and are commonly used for the set up of metastatic rat tibia bone cancer pain models. We constructed a model of bone cancer pain induced by the injection of Walker 256 carcinoma cells into the tibia cavity of rats, as the research basis of our study.
Some structures in brainstem could elicit desending signals to spinal cord regulate pain signal transmission to central nervous system, and it is one of the superspinal pain medulating structures, which are involved in pain signal integration and modulation. So, to stop the superspinal nociceptive signal up or down transmission could be a novel method for pain management. Rostral ventromedial medulla (RVM) play a critical role in the brainstem descending facilitation system. RVM contains nucleus like 5-HT abundant nucleus raphe magnus (NRM), the adjacent gigantocellularis pars alpha and ventral nucleus reticularis gigantocelluaris. RVM receives the nociceptive information from midbrain periaqueductal gray (PAG), in turn projects to the spinal cord largely along the dorsolateral funiculus (DLF) and terminate in the spinal cord. The PAG, RVM with its spinal projections, constitute the channel of the descending pain control system, which could facilitate or inhibit spinal transmission of pain signal. The ablation of RVM facilitory neurons might attenuate the pain transmission, and thus represent a novel method for pain management.
Intra-RVM microinjection of Lidocaine was used to block the function of facilitory neurons in RVM in a rat model of neuropathic pain. The analgesic effects were significant, but as the metabolism of the drug, analgesic effects decreased and finally disappeared. Intra-RVM injection of saporin, which is a neurotoxin, destroyed the RVM neurons and could eventually produce a long-term analgesic effect. However, the necrosis of neurons of RVM could lead to neuroimmune and/or neuroinflammatory responses of adjacent neural tissue. RVM is implicated in multiple important physiological functions, such as respiratory, heart rate, blood pressure, thermoregulation and motion function, the side effects induced from the products of necrosis could be severe and mortal.
Caspases3, one of the most downstream caspases, plays a key role in executing programmed cell death, but the activation of wild caspase3 depends on exogenous stimulations and a long procedure of the activation of predomain caspases. A new human recombinant caspase3 was generated by Srinivasula in a reverse order of the big and small subunits by molecular biology methods. Unlike the wild caspase3, this recombinant caspase3 could induce constitutive apoptosis without exogenous stimulations and the activation of predomain caspases. This recombinant caspase3 gene could be used as a novel cell death factor in the genetic engineering. However, as a hetergenetic gene, the human reconstitutive caspase3 gene could induce immunoreactions in experimentary rats. In this study, we will generate rat recombinant caspase3 gene, and inject this self-activated gene with non-viral vector PEI into the RVM in a rat model of metastatic tibia bone cancer pain. Rat recombinant caspase3 could induce neuron apotosis in the RVM of bone cancer pain rats without immuo or inflammatory responses, but produce a long-term analgesic effect against bone cancer pain.
Methods and Results
1、Construction of rat constitutively active recombinant caspase3 gene and investigation of its pro-apoptotic effect in vitro
Methods:Total RNA of rat astroglioma C6 cells was extracted and the reverse transcription PCR was performed to gain cDNA. According to Genbank, the primers of large and small subunits of wild caspase3 were designed and reversed rat caspase3 gene was gained by settling the small subunit prior to the large one through recombinant PCR, and cloned into the expression vector pcDNA3.1+-Remcasp3 and C1-Remcasp3 to transfect human 293T cells and rat immortalized neural progenitor cells (INPC). The expression and pro-apoptotic effect of recombinant caspase-3 was observed by the changes of morphology of the transfected cells through immunofluorescence and analyzed by Annexin V-FITC staining, Flowcytometry and MTT assay. Transmission electron microscope was used to detect the micro morphology changes of apoptosis of the transfected cell.
Results:The rat recombinant caspase3 with reversed large and small subunits was successfully contructured. The sequencing result was the same as designed. After transfected with C1-Remcasp3, INPC and 293T cells presented obvious apoptosis. Transmission electron microscope detected cytoplasm concentration, chromatin condensation and the formation of apoptotic bodies. MTT assay showed that the proliferation of recombinant caspase-3 transfected cells was significantly inhibited(INPC: 44.61±0.15%; 293T:48.35±0.16%) (P<0.05).Annexin V-FITC staining revealed that the percentage of apoptotic cells in the transfectants of recombinant caspase3 gene was(INPC: 16.00±0.99%; 293T:30.67±1.52%),which was much higher than that of control cells (P<0.05)
2、Contruction and evaluation of the rat model of bone cancer pain
Methods:Cultured rat Walker 256 mammary gland carcinoma cells was injected into the rat abdominal cavity.6-7 days after injection, the rat peritoneal fluid was extracted and prepared for tibia injection. Thirty-two female SD rats were divided into 4 groups at random,8 rats in each group:ⅠNaive group;ⅡD-Hank's group:10μl sterilized D-Hank's solution was injected into the tibia cavity of the right leg of rats; III Heat-killed cells group: 10μl heat-killed Walker 256 carcinoma cell solution was injected into the tibia cavity of the right leg of rats, and IV Bone cancer pain group:10μl live Walker 256 carcinoma cell solution was injected into the tibia cavity of the right leg of rats. Mechanical allodynia, mechanical hyperalgesia and ambulatory pain were measured one day before sugery, the day of surgery, and each day to the 15th day after sugery. X ray was used on the 15th day after sugery. After radiological detection, the rats were sacrificed and the GFAP and OX42 immunofluorescence were used and high performance liquid chromatography (HPLC) method was taken to measure the glutamate concentrations in RVM and spinal cord.
Results:Significant behavioral changes were observed in the rats of BCP group:the mechanical withdrawal threshold and latency were significantly decreased, as compared with other groups.15 days after injection with live carcinoma cells, tibia bone destruction was monitored using radiological methods and showed the signs of radiolucent and deterioration with medullary bone loss, close to the injection site, While No radiological changes were observed on the normal tibia bone. In the immunofluoresence test, GFAP and OX42 were used to label the astrocytes and microglia in spinal cord. In bone cancer pain group, the number of immunoreactive astrocytes and microglia were significantly increased, and fluorescence intensity were much higher than the other control groups. The results of high performance liquid chromatography showed that the concentration of Glutamate in RVM and spinal cord were significantly higher than the other control groups (P<0.05).
3. Analgesic effects of targeted intra-RVM injection of In-vivo jetPEIin a rat model of bone cancer pain
Methods:After being identified, the plasmid pcDNA3.1+-Remcasp3 was prepared in large scale. The concentration and purity were measured. The preparation of the in-vivo jetPEI/DNA complexes were performed under sterile conditions:the DNA and non-viral vector were mixed under certain proportion and added into sterilized 10% glucose solution. Sterile water was added into the mixture to obtain a final concentration of 5% glucose. 10～12 days after intra tibia injection of carcinoma cells, rats were anesthetized with propofol (50μg/100g i.p.). Then they were positioned in a stereotaxic apparatus and a single microinjection into RVM was performed according to its anatomical position (anteroposterior,-11.0 mm from bregma; lateral,±0.6mm from midline; dorsoventral,-8.5 mm from the cranium). Drug administration into the RVM was performed by slowly and carefully expelling 1μl of the solution into RVM for both sides. The needle was left in position for a minute to allow sufficient diffusion of drugs before the needle was withdrawn. Behavioral tests were taken each day on one day before intra-RVM injection, the day after injection, to the 7th day after intracranial injection.48h after intra-RVM injection of DNA/PEI mixure, rats were sacrificed and paraffin sections of RVM were prepared for TUNEL detection. Spinal cord GFAP and OX42 immunofluorescence tests were taken and high performance liquid chromatography (HPLC) method was taken to measure the glutamate concentrations in RVM and spinal cord.
Results:Before intraRVM injection, there were no significant differences in withdrawal thresholds, withdrawal duration and score of ambulatory pain among different groups of rats.24h after intraRVM injection of recombinant caspase3 and PEI mixture, the withdrawal thresholds were significantly increased, withdrawal durations and the score of ambulatory pain were significantly down regulated (P<0.05). A lot of apoptotic cells were found in RVM. The concentration of glutamate in RVM and spinal cord of BCP rats were significantly down regulated after intraRVM injection of Remcasp3/PEI mixture, as compared with BCP rats without intraRVM injection of recombinant caspase3 (P<0.05). In the immunofluoresence test, the number of immunoreactive astrocytes and microglia were significantly increased in bone cancer pain group of rats. After treated with recombinant caspase3 and PEI mixture in RVM, the number of activated glial cells decreased, and the level of both astrocytes and microglia activation were significantly down-regulated.
4. Safty evaluation of targeted intraRVM injection of Recombinant caspase3/in-vivo jetPEI mixture
Methods:From one day before intraRVM injection, the day of the injection, to 7th day after surgery, the basic physiological function parameters were measured in Naive group, PEI group,5%Glucose group, PEI+5%Glucose group and Remcasp3 group of rats. The weight of body, respiratory rate, rectal temperature, and heart rate were measured.
Results:There were no significant differences on physiological parameters before intra RVM injection. After RVM injection, the body weight, rectal temperature, respiratory rates and heart rates were lack of significant differences among 5 groups of rats in the whole observation time(P>0.05).
5. Statistical analysis
All of the analyses were performed by SigmaStat 3.0 software package. All data were expressed as the mean±standard deviation (SD). Group comparisons and the other results using one-way analysis of variance. P-value of< 0.05 was considered statistically significant.
Summary
1. Major results
(1) The rat recombinant caspase3 can be expressed in eukaryocytes, and induce apoptosis without exogenous stimulation.
(2) IntraRVM injection of In-vivo jetPEI/recombinant caspase3 mixture could induce apoptosis to the neurons in RVM and produce significant, complete and long lasting analgesic effects in bone cancer pain rats.
(3) The concentration of excitatory amino acids-Glutamate in the center nervous system and the number of activated astrocytes and microgial cells in spinal cord of bone cancer pain rats are significantly higher than normal rats. After inducing apoptosis into the neurons in RVM, the concentrations of glutamate and the activation of glial cells were significantly down regulated.
(4) A single intraRVM injection and inducing apoptosis to neurons of RVM could not produce significant changes in the motor and basic physiological functions of experiment rats.
2. Conclusions
(1) The rat recombinant caspase3 was successfully constructed in-vitro, and could be expressed in eukaryocytes. It can induce constitutely self apoptosis to neural and non-neural cells without sitmulaitons or predomain caspases. It could be used safely in rats with normal immunosystem and provide a new "cell death" gene for the research and management of gene therapy for central nervous system diseases.
(2) Similar to neuropathic pain and inflammatory pain, RVM is critical in the initaion and mantance of bone cancer pain. Targeted ablation of the neurons in RVM by means of apoptosis could produce effective and safe analgesic effects.
(3) Non-viral catonic PEI could be used as a high effective and safe vector for central nervous system transfection.
(4) The level of excitatory amino acids glutamate and the activation of spinal glial cells are closed involved in the intiation and maintance of bone cancer pain.
(5) Stereotactic microinjection of DNA/PEI mixture into RVM did not induce significant changes to the physiological functions of experiment animals. This suggested that targeted ablation of RVM could produce effective, long lasting and safe analgesic effects and subsequently provide us a new approach for superspinal pain management targets.
癌痛的治疗一直是临床上的难题,随着对恶性肿瘤的诊断和治疗技术的不断进步,癌性疼痛已经成为严重影响患者生活质量和生存率的主要问题。75%-90%的转移性恶性肿瘤患者会发生严重的癌性疼痛。恶性肿瘤,如前列腺癌,乳腺癌和肺癌等,均易发生骨转移,引起病理性骨痛,即骨癌痛。骨癌痛性质严重,而且发生率极高。目前对于骨癌痛的治疗主要依赖阿片类药物,但随着时间的延长,其用量逐渐增大,并产生严重的副作用,如成瘾,耐受,恶心,呕吐,便秘和呼吸抑制等。因此极有必要寻求一种基于疼痛机制性的,具有高效、作用持久、毒副作用小等优点的新的治疗方案。
骨癌痛动物模型的建立为研究者提供了研究骨癌痛复杂机制和探索更有效靶向药物治疗的契机,特别是大鼠胫骨转移性癌痛模型,其与人类骨癌痛的发生机制有诸多相似之处,能较好的模拟人类转移性骨癌痛的发生,具有重大的研究意义。在此实验模型中,侵袭性肿瘤细胞被注射到大鼠股骨或胫骨骨髓腔。随着肿瘤的生长和骨质破坏,可产生一系列行为学,细胞学和神经生化学改变。Walker256乳腺癌细胞具有明显的骨侵袭能力,常用于建立大鼠转移性胫骨癌痛模型。因此,本研究拟采用Walker256乳腺癌细胞建立大鼠胫骨转移癌痛模型,为下一步的研究奠定基础。
脑干中的某些结构会向脊髓发出“下行，，信号,调控来自脊髓背角的痛觉信息向中枢的传递,并参于各种痛觉相关信息的整合和处理,是疼痛调节的高级中枢。因此,在脊髓上水平完全阻断伤害性信号的上行或下行性传导,有望成为治疗顽固性疼痛的理想策略。延髓头端腹内侧区(rostral ventromedial medulla, RVM)是脑干下行调控系统的重要结构,其内有富含5—羟色胺的中缝大核(nucleus raphe magnus, NRM),网状巨细胞核α部(gigantocellularis pars alpha)和外侧网状巨细胞核等神经核团。RVM区接受并整合来自中脑导水管周围灰质(periaqueductal gray, PAG)的信息,其内神经元轴突沿脊髓背侧束(dorsolateral funiculus, DLF)下行并终于脊髓,构成了疼痛信号下行调控系统的主要输出通路,并能易化或抑制脊髓痛觉信号传递。因此,毁损RVM区易化神经元是削弱慢性疼痛下行易化调控系统作用,治疗顽固性疼痛的有效方法。
以往有研究者利用颅内微注射的方法,在慢性疼痛模型大鼠RVM区内注射利多卡因封闭其易化神经元的功能,虽然达到了良好的镇痛效果,但随着药物代谢,其镇痛作用逐渐消失；也有研究者将神经毒素—皂角素注射到慢性疼痛模型大鼠的RVM区,毁损RVM区神经元,这种方法虽然可以获得持久的镇痛效果,但由于神经元坏死,不可避免的会诱发周围神经组织炎性免疫反应。RVM区调控机体呼吸,心率,血压,体温,运动等重要生理功能。神经元坏死产生的化学物质所诱发的炎性及免疫反应,很可能对机体正常生理功能产生严重的影响。
Caspase 3是多数细胞凋亡途径中的末端效应蛋白酶,但其活化需要外源刺激及上游一系列caspase分子级联反应。Srinivasula等通过分子生物学的方法模拟caspase3在细胞内的活化过程,将其大、小亚基序列颠倒后,构建了无需外源刺激或上游caspase分子作用,就可自发活化的人源性重组caspase3基因,直接介导靶细胞凋亡,可作为基因治疗领域一个极好的细胞致死基因。但人源性基因可能会诱发实验大鼠免疫反应,干扰实验结果的准确性,因此,本研究拟通过构建大鼠源性重组Caspase3基因,并将其与非病毒载体PEI共同定向微注射到胫骨转移癌痛模型大鼠RVM区,启动RVM区神经元自发凋亡,达到“永久”性治疗骨癌痛的效果。
研究方法与结果
1、大鼠源性重组caspase3基因的构建及促凋亡效应评价
方法：提取大鼠星形胶质瘤C6细胞总RNA后,通过逆转录PCR的方法得到cDNA。根据Genbank中caspase3基因的序列分别设计其大、小亚基的引物,并通过重组PCR的方法获得大小亚基顺序颠倒的大鼠源性重组caspase3基因(Remcasp3)。将Remcasp3分别定向克隆到真核表达载体pcDNA3.1+和C1后分别转染人胚肾293T细胞和大鼠永生化神经干细胞,观察细胞形态学变化,通过Annexin V-PI双染、流式细胞术、MTT法来检测其促细胞凋亡作用,透射电镜检测其凋亡微形态学改变。
结果：成功构建出大小亚基顺序颠倒的大鼠源性重组caspase3基因片段,测序结果与预期结果相一致。转染C1-Remcasp3后,INPC及293T出现明显凋亡细胞。电镜下可见胞质浓缩,核染色质边集,并有凋亡小体的形成等典型的细胞凋亡的特征。MTT检测结果提示两种细胞的增殖明显被抑制(INPC:44.61±0.15%; 293T:48.35±0.16%),与对照组相比有统计学差异(P<0.05)。Annexin V-PI共染色流式细胞术检测两种细胞的凋亡率(INPC:16.002±0.99%; 293T:30.667±1.52%),与对照组相比有统计学差异(P<0.05)。
2、大鼠骨癌痛模型的构建及评价
方法：将培养的Walker 256大鼠乳腺癌细胞注入大鼠腹腔内。接种6-7d后收集癌性腹水,经处理后用于胫骨接种。雌性SD大鼠32只,随机分为4组,每组8只。Ⅰ为正常对照组；Ⅱ为D-Hank's组：将10μl无菌D-Hank's溶液注射至大鼠右侧胫骨上段骨髓腔；Ⅲ为热灭活细胞组：于大鼠右侧胫骨上段骨髓腔注射热灭活的Walker256乳腺癌细胞悬液10μl；Ⅳ为骨癌痛模型组：于大鼠右侧胫骨上段骨髓腔注射有活性的Walker256乳腺癌细胞悬液10μl。分别于模型制备前一天,当天,至术后第十五天,每天检测机械性痛觉超敏,机械性痛觉过敏和移动诱发痛,并于模型制备后第15天进行后肢X射线检查。放射学检查后,分别进行大鼠脊髓GFAP,OX42免疫荧光染色和脑干RVM区及脊髓匀浆高效液相色谱(high performance liquid chromatography, HPLC)检测中枢神经系统谷氨酸的浓度。
结果：骨癌痛模型组大鼠出现了明显的痛觉行为学改变,与对照组相比差异有统计学意义(P＜0.05)。接种了有活性的肿瘤细胞后第十五天进行放射学检查,在术侧胫骨注射部位及周围出现较严重的骨质破坏,骨皮质缺失,而对照组大鼠术侧骨皮质完好,未发现骨质破坏。激光共聚焦检测大鼠脊髓GFAP,OX42免疫荧光发现,骨癌痛组大鼠GFAP标记的星形胶质细胞和OX42标记的小胶质细胞明显活化,胞体增大,数量增多。高效液相色谱结果显示：骨癌痛组大鼠RVM区和脊髓兴奋性氨基酸谷氨酸含量均显著升高,与对照组相比差异有统计学意义(P＜0.05)。
3、RVM区靶向注射In-vivo jetPEI/重组caspase3基因复合物在骨癌痛大鼠的镇痛效应评价
方法：鉴定pcDNA3.1+-Remcasp3单克隆菌种,扩增后进行性质粒大量制备,并计算质粒DNA的纯度和浓度。在无菌条件下将质粒DNA与非病毒载体In-vivo jetPEI按一定比例混匀后溶于10%无菌葡萄糖溶液中,再加入无菌双蒸水使葡萄糖的终浓度为5%。在实验大鼠接种有活性的肿瘤细胞10～12天后,以丙泊酚麻醉实验大鼠(50μg/100g i.p.),置于脑立体定位仪上,依据RVM区的解剖位置(前后位：距前囟11mm；左右位：距正中线各旁开0.6mm；背腹位：距颅骨8.5mm)。将药物缓慢注射入双侧RVM区(左右各1μl)。停留片刻后,缓慢退出微量加样器。分别在立体定位注射前一天,当天,至手术后第七天,每天进行痛觉行为学检测。立体定位注射DNA/PEI混合物后48h,分别取Remcasp3组和正常对照组大鼠RVM区,制备组织块石蜡切片,进行Tunel检测；制备脊髓切片进行GFAP,OX42免疫荧光染色；取脑干RVM区及脊髓匀浆以高效液相色谱(HPLC)法检测中枢神经系统谷氨酸的浓度。
结果：RVM区内微注射前,各组骨癌痛大鼠机械缩爪阈值,缩爪反应时间和移动诱发痛觉评分无统计学差异。RVM区内立体定位注射重组质粒／PEI混合物24h后,大鼠机械性缩爪阈值明显升高,缩爪反应时间明显缩短,移动诱发痛觉评分显著降低,差异有统计学意义(P<0.05)。Tunel检测结果显示RVM区可见大量凋亡细胞。HPLC检测结果显示骨癌痛组大鼠RVM区和脊髓内谷氨酸含量在靶向注射重组自杀基因／PEI复合物后显著下降。与癌痛组相比,差异有统计学意义(P＜0.05)。在免疫荧光实验中,骨癌痛组大鼠GFAP标记的星形胶质细胞和OX42标记的小胶质细胞明显活化,数量增多,而颅内微注射重组质粒／PEI复合物后,星形胶质细胞和小胶质细胞的活化明显受到抑制,活化细胞数量显著减少。
4、RVM区内靶向注射重组caspase3/In-vivo jetPEI的安全性评价
方法：分别于RVM区微注射前,及手术后当天,第1天,第2天,第3天至第7天分别测量Naive组,PEI组,5%Glucose组,PEI+5%Glucose组和Remcasp3组大鼠体重,直肠温度,呼吸频率和心率值。
结果：术前各组大鼠生理参数基础值无统计学差异,RVM区微注射后整个观察期内,各组大鼠体重、直肠温度、呼吸频率、心率等生命体征参数之间均无显著差异(P＞0.05)。
5、统计学处理
采用SigmaStat 3.0统计软件进行处理。计量资料以均数±标准差((?)±s)表示,行为学测定及其余计量资料采用单因素方差分析。P＜0.05为差异有统计学意义。
研究总结
一主要研究结果
1、大鼠源性重组caspase3基因可在真核细胞中表达,并在无外源性刺激下直接诱导细胞发生凋亡。
2、RVM区内注射In-vivo jetPEI/重组caspase3基因复合物能诱发其神经元凋亡,并可对骨癌痛大鼠产生显著,完善,持久的镇痛效果。
3、骨癌痛大鼠中枢神经系统兴奋性氨基酸谷氨酸含量显著升高,脊髓星形胶质细胞,小胶质细胞明显活化,数量增多；诱导RVM区神经元凋亡后,中枢神经系统谷氨酸水平显著下降,脊髓星形胶质细胞,小胶质细胞活化水平显著下降。
4、RVM区单次注射,并且诱导其内神经元凋亡不会对实验大鼠的运动功能和生理功能产生显著的影响。
二、研究结论
1、本研究构建的大鼠源性重组caspase3基因能够在真核细胞中表达,并在无外源刺激和上游活化的情况下直接诱导神经细胞与非神经细胞自发凋亡,为基于免疫功能正常的实验大鼠相关疾病的研究和治疗提供一种新型有效的细胞杀伤基因,并为神经系统难治性疾病的基因治疗提供了又一种有效的目的基因。
2、与神经病理性痛和炎性痛的发生机制相似,RVM区在骨癌痛的产生和维持中也起到重要作用。以凋亡的方式毁损RVM区易化神经元能够起到有效并且安全的镇痛作用。
3、非病毒阳离子载体PEI能将目的基因安全,高效地转染至中枢神经系统。
4、中枢神经系统兴奋性氨基酸谷氨酸水平,脊髓星形胶质细胞,小胶质细胞的活化状态与骨癌痛的发生发展显著相关。
5、立体定位引导下RVM区单次注射DNA/PEI混合物未对实验动物的生理功能产生明显影响,提示RVM核团的靶向毁损可以发挥有效、持久和完善的镇痛效果,且具有较高的安全性,为顽固性疼痛治疗提供了一个有效的脊髓上治疗靶点。
Background
Cancer-related pain has always been a difficult clinical issue. As great improvements have been made in the detection and treatment of most types of cancer, cancer pain have played a significant negative role in the quality of lives and the survival rates of cancer patients. Between 75 and 90% of patients with metastatic cancer will experience significant cancer-induced pain. Cancers, including prostate cancer, breast cancer, and lung cancer, have special affinities to metastasize to bone, and produce pathological bone cancer pain. Bone cancer pain is one of the most severe cancer induced pain with high morbidity. The treatment of bone cancer pain now has been largely based on the use of opioids. Opioids are effective in attenuating bone cancer induced pain, but with the increases in frequence and doses, the analgesic effects from opioids are frequently accompanied by side effects such as addiction, tolerance, nausea, vomiting, constipation and respiratory depression. Therefore, it is very necessary to explore a novel mechanism-based treatment which has more effective, long-lasting and less side-effects to relieve the bone cancer pain.
The recently published animal models of bone cancer pain have allowed the researchers greater insights into the complicated mechanisms of bone cancer pain and the explore more effective targeted drug managements. The metastatic rat tibia bone cancer pain presents the similar characteristic pain symptoms to human bone cancer pain. In this model, metastative cancer cells were injected into the intramedullary cavity of femur or tibia of rats to produce a serie of behavioral, cellular, and neurochemical changes correlated with the development of cancer growth and bone destruction. Walker 256 carcinoma cells have significant bone metastatic affinities, and are commonly used for the set up of metastatic rat tibia bone cancer pain models. We constructed a model of bone cancer pain induced by the injection of Walker 256 carcinoma cells into the tibia cavity of rats, as the research basis of our study.
Some structures in brainstem could elicit desending signals to spinal cord regulate pain signal transmission to central nervous system, and it is one of the superspinal pain medulating structures, which are involved in pain signal integration and modulation. So, to stop the superspinal nociceptive signal up or down transmission could be a novel method for pain management. Rostral ventromedial medulla (RVM) play a critical role in the brainstem descending facilitation system. RVM contains nucleus like 5-HT abundant nucleus raphe magnus (NRM), the adjacent gigantocellularis pars alpha and ventral nucleus reticularis gigantocelluaris. RVM receives the nociceptive information from midbrain periaqueductal gray (PAG), in turn projects to the spinal cord largely along the dorsolateral funiculus (DLF) and terminate in the spinal cord. The PAG, RVM with its spinal projections, constitute the channel of the descending pain control system, which could facilitate or inhibit spinal transmission of pain signal. The ablation of RVM facilitory neurons might attenuate the pain transmission, and thus represent a novel method for pain management.
Intra-RVM microinjection of Lidocaine was used to block the function of facilitory neurons in RVM in a rat model of neuropathic pain. The analgesic effects were significant, but as the metabolism of the drug, analgesic effects decreased and finally disappeared. Intra-RVM injection of saporin, which is a neurotoxin, destroyed the RVM neurons and could eventually produce a long-term analgesic effect. However, the necrosis of neurons of RVM could lead to neuroimmune and/or neuroinflammatory responses of adjacent neural tissue. RVM is implicated in multiple important physiological functions, such as respiratory, heart rate, blood pressure, thermoregulation and motion function, the side effects induced from the products of necrosis could be severe and mortal.
Caspases3, one of the most downstream caspases, plays a key role in executing programmed cell death, but the activation of wild caspase3 depends on exogenous stimulations and a long procedure of the activation of predomain caspases. A new human recombinant caspase3 was generated by Srinivasula in a reverse order of the big and small subunits by molecular biology methods. Unlike the wild caspase3, this recombinant caspase3 could induce constitutive apoptosis without exogenous stimulations and the activation of predomain caspases. This recombinant caspase3 gene could be used as a novel cell death factor in the genetic engineering. However, as a hetergenetic gene, the human reconstitutive caspase3 gene could induce immunoreactions in experimentary rats. In this study, we will generate rat recombinant caspase3 gene, and inject this self-activated gene with non-viral vector PEI into the RVM in a rat model of metastatic tibia bone cancer pain. Rat recombinant caspase3 could induce neuron apotosis in the RVM of bone cancer pain rats without immuo or inflammatory responses, but produce a long-term analgesic effect against bone cancer pain.
Methods and Results
1、Construction of rat constitutively active recombinant caspase3 gene and investigation of its pro-apoptotic effect in vitro
Methods:Total RNA of rat astroglioma C6 cells was extracted and the reverse transcription PCR was performed to gain cDNA. According to Genbank, the primers of large and small subunits of wild caspase3 were designed and reversed rat caspase3 gene was gained by settling the small subunit prior to the large one through recombinant PCR, and cloned into the expression vector pcDNA3.1+-Remcasp3 and C1-Remcasp3 to transfect human 293T cells and rat immortalized neural progenitor cells (INPC). The expression and pro-apoptotic effect of recombinant caspase-3 was observed by the changes of morphology of the transfected cells through immunofluorescence and analyzed by Annexin V-FITC staining, Flowcytometry and MTT assay. Transmission electron microscope was used to detect the micro morphology changes of apoptosis of the transfected cell.
Results:The rat recombinant caspase3 with reversed large and small subunits was successfully contructured. The sequencing result was the same as designed. After transfected with C1-Remcasp3, INPC and 293T cells presented obvious apoptosis. Transmission electron microscope detected cytoplasm concentration, chromatin condensation and the formation of apoptotic bodies. MTT assay showed that the proliferation of recombinant caspase-3 transfected cells was significantly inhibited(INPC: 44.61±0.15%; 293T:48.35±0.16%) (P<0.05).Annexin V-FITC staining revealed that the percentage of apoptotic cells in the transfectants of recombinant caspase3 gene was(INPC: 16.00±0.99%; 293T:30.67±1.52%),which was much higher than that of control cells (P<0.05)
2、Contruction and evaluation of the rat model of bone cancer pain
Methods:Cultured rat Walker 256 mammary gland carcinoma cells was injected into the rat abdominal cavity.6-7 days after injection, the rat peritoneal fluid was extracted and prepared for tibia injection. Thirty-two female SD rats were divided into 4 groups at random,8 rats in each group:ⅠNaive group;ⅡD-Hank's group:10μl sterilized D-Hank's solution was injected into the tibia cavity of the right leg of rats; III Heat-killed cells group: 10μl heat-killed Walker 256 carcinoma cell solution was injected into the tibia cavity of the right leg of rats, and IV Bone cancer pain group:10μl live Walker 256 carcinoma cell solution was injected into the tibia cavity of the right leg of rats. Mechanical allodynia, mechanical hyperalgesia and ambulatory pain were measured one day before sugery, the day of surgery, and each day to the 15th day after sugery. X ray was used on the 15th day after sugery. After radiological detection, the rats were sacrificed and the GFAP and OX42 immunofluorescence were used and high performance liquid chromatography (HPLC) method was taken to measure the glutamate concentrations in RVM and spinal cord.
Results:Significant behavioral changes were observed in the rats of BCP group:the mechanical withdrawal threshold and latency were significantly decreased, as compared with other groups.15 days after injection with live carcinoma cells, tibia bone destruction was monitored using radiological methods and showed the signs of radiolucent and deterioration with medullary bone loss, close to the injection site, While No radiological changes were observed on the normal tibia bone. In the immunofluoresence test, GFAP and OX42 were used to label the astrocytes and microglia in spinal cord. In bone cancer pain group, the number of immunoreactive astrocytes and microglia were significantly increased, and fluorescence intensity were much higher than the other control groups. The results of high performance liquid chromatography showed that the concentration of Glutamate in RVM and spinal cord were significantly higher than the other control groups (P<0.05).
3. Analgesic effects of targeted intra-RVM injection of In-vivo jetPEIin a rat model of bone cancer pain
Methods:After being identified, the plasmid pcDNA3.1+-Remcasp3 was prepared in large scale. The concentration and purity were measured. The preparation of the in-vivo jetPEI/DNA complexes were performed under sterile conditions:the DNA and non-viral vector were mixed under certain proportion and added into sterilized 10% glucose solution. Sterile water was added into the mixture to obtain a final concentration of 5% glucose. 10～12 days after intra tibia injection of carcinoma cells, rats were anesthetized with propofol (50μg/100g i.p.). Then they were positioned in a stereotaxic apparatus and a single microinjection into RVM was performed according to its anatomical position (anteroposterior,-11.0 mm from bregma; lateral,±0.6mm from midline; dorsoventral,-8.5 mm from the cranium). Drug administration into the RVM was performed by slowly and carefully expelling 1μl of the solution into RVM for both sides. The needle was left in position for a minute to allow sufficient diffusion of drugs before the needle was withdrawn. Behavioral tests were taken each day on one day before intra-RVM injection, the day after injection, to the 7th day after intracranial injection.48h after intra-RVM injection of DNA/PEI mixure, rats were sacrificed and paraffin sections of RVM were prepared for TUNEL detection. Spinal cord GFAP and OX42 immunofluorescence tests were taken and high performance liquid chromatography (HPLC) method was taken to measure the glutamate concentrations in RVM and spinal cord.
Results:Before intraRVM injection, there were no significant differences in withdrawal thresholds, withdrawal duration and score of ambulatory pain among different groups of rats.24h after intraRVM injection of recombinant caspase3 and PEI mixture, the withdrawal thresholds were significantly increased, withdrawal durations and the score of ambulatory pain were significantly down regulated (P<0.05). A lot of apoptotic cells were found in RVM. The concentration of glutamate in RVM and spinal cord of BCP rats were significantly down regulated after intraRVM injection of Remcasp3/PEI mixture, as compared with BCP rats without intraRVM injection of recombinant caspase3 (P<0.05). In the immunofluoresence test, the number of immunoreactive astrocytes and microglia were significantly increased in bone cancer pain group of rats. After treated with recombinant caspase3 and PEI mixture in RVM, the number of activated glial cells decreased, and the level of both astrocytes and microglia activation were significantly down-regulated.
4. Safty evaluation of targeted intraRVM injection of Recombinant caspase3/in-vivo jetPEI mixture
Methods:From one day before intraRVM injection, the day of the injection, to 7th day after surgery, the basic physiological function parameters were measured in Naive group, PEI group,5%Glucose group, PEI+5%Glucose group and Remcasp3 group of rats. The weight of body, respiratory rate, rectal temperature, and heart rate were measured.
Results:There were no significant differences on physiological parameters before intra RVM injection. After RVM injection, the body weight, rectal temperature, respiratory rates and heart rates were lack of significant differences among 5 groups of rats in the whole observation time(P>0.05).
5. Statistical analysis
All of the analyses were performed by SigmaStat 3.0 software package. All data were expressed as the mean±standard deviation (SD). Group comparisons and the other results using one-way analysis of variance. P-value of< 0.05 was considered statistically significant.
Summary
1. Major results
(1) The rat recombinant caspase3 can be expressed in eukaryocytes, and induce apoptosis without exogenous stimulation.
(2) IntraRVM injection of In-vivo jetPEI/recombinant caspase3 mixture could induce apoptosis to the neurons in RVM and produce significant, complete and long lasting analgesic effects in bone cancer pain rats.
(3) The concentration of excitatory amino acids-Glutamate in the center nervous system and the number of activated astrocytes and microgial cells in spinal cord of bone cancer pain rats are significantly higher than normal rats. After inducing apoptosis into the neurons in RVM, the concentrations of glutamate and the activation of glial cells were significantly down regulated.
(4) A single intraRVM injection and inducing apoptosis to neurons of RVM could not produce significant changes in the motor and basic physiological functions of experiment rats.
2. Conclusions
(1) The rat recombinant caspase3 was successfully constructed in-vitro, and could be expressed in eukaryocytes. It can induce constitutely self apoptosis to neural and non-neural cells without sitmulaitons or predomain caspases. It could be used safely in rats with normal immunosystem and provide a new "cell death" gene for the research and management of gene therapy for central nervous system diseases.
(2) Similar to neuropathic pain and inflammatory pain, RVM is critical in the initaion and mantance of bone cancer pain. Targeted ablation of the neurons in RVM by means of apoptosis could produce effective and safe analgesic effects.
(3) Non-viral catonic PEI could be used as a high effective and safe vector for central nervous system transfection.
(4) The level of excitatory amino acids glutamate and the activation of spinal glial cells are closed involved in the intiation and maintance of bone cancer pain.
(5) Stereotactic microinjection of DNA/PEI mixture into RVM did not induce significant changes to the physiological functions of experiment animals. This suggested that targeted ablation of RVM could produce effective, long lasting and safe analgesic effects and subsequently provide us a new approach for superspinal pain management targets.
引文
1. Andrea Doseff. Apoptosis:The Sculptor of Development. Stem Cells and Development 2004, 13:473-483
2. Savill J. Apoptosis in resolution of inflammation. J Leuk Biol 1997,61:375-380.
3. Ting-Jun FAN, Li-Hui HAN, Ri-Shan CONG et al. Caspase Family Proteases and Apoptosis. Acta Biochimica et Biophysica Sinica 2005,37(11):719-727.
4. Shi Y. Caspase activation, inhibition, and reactivation:a mechanistic view. Protein Sci, 2004,13(8):1979-1987.
5. Fadeel B and Orrenius S. Apoptosis:a basic biological phenomenon with wide-ranging implications in human disease. J Intern Med,2005,258(6):479-517.
6. Hengartner MO. The biochemistry of apoptosis. Nature 2000,407(6805):770-776.
7. Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol,2004,5(11):897-907.
8. Srinivasula SM, Ahmad M, MacFarlane M, et al. Generation of constitutively active recombinant caspases-3 and-6 by rearrangement of their subunits. J Biol Chem,1998, 273(17):10107-10111.
9. Fischer U and Schulze-Osthoff K. Apoptosis-based therapies and drug targets. Cell Death Differ,2005,12 Suppl 1:942-961.
10. Chelur DS and Chalfie M. Targeted cell killing by reconstituted caspases. Proc Natl Acad Sci U S A,2007,104(7):2283-2288.
11. Jia LT, Zhang LH, Yu CJ, et al. Specific tumoricidal activity of a secreted proapoptotic protein consisting of HER2 antibody and constitutively active caspase-3. Cancer Res,2003, 63(12):3257-3262.
12. Chelur DS and Chalfie M. Targeted cell killing by reconstituted caspases. Proc Natl Acad Sci U S A,2007,104(7):2283-2288.
13.高峰,田玉科,杨辉等.猿肾病毒SV40大T抗原基因永生化大鼠神经前体细胞株的构建.中华麻醉学杂志,2005,25：597-600.
14. Hengartner MO. The biochemistry of apoptosis. Nature,2000,407(6805):770-776
[1]Slavin KV, Tesoro EP, Mucksavage JJ. The treatment of cancer pain. Drugs Today (Barc) 2004;40:235-45. Biology of Bone Cancer Pain Michael J. Goblirsch, Pawel P. Zwolak, and Denis R. Clohisy Clin Cancer Res 2006;12(20 Suppl) October 15,2006.
[2]Bone Cancer Pain:From Model to Mechanism to Therapy. Nancy M. Luger, BA, David B. Mach, BS, Molly A. Journal of Pain and Symptom Management Vol.29 No.5S May 2005
[3]Cherny NI. The management of cancer pain. Ca Cancer J Clin 2000;50:70-116. quiz 117-120.
[4]Koshy RC, Grossman SA. Treatment of bone pain. In:Body JJ, ed. Tumor bone diseases and osteoporosis in cancer patients. New York:Marcel Dekker, Inc,2000:245-261.
[5]Medhurst SJ, Walker K, Bowes M, et al. A rat model of bone cancer pain. Pain,2002, 96(1-2):129-140.
[6]Mao-Ying QL, Zhao J, Dong ZQ et al. A rat model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells[J]. Biochem Biophys Res Commun,2006,345(4):1292-1298.
[7]Walker 256 Tumor-Bearing Rats as a Model to Study Cancer Pain. Patricia Brigatte, Sandra Coccuzzo Sampaio, Vanessa Pacciari Gutierrez et al. The Journal of Pain, Vol 8, No 5 (May),2007:pp 412-421
[8]Chaplan SR, Bach FW, Pogrel JW et al. Quantitative assessment of tactile allodynia in the rat paw[J]. J Neurosci Methods,1994,53(1):55-63.
[9]Decosterd I, Woolf CJ. Spared nerve injury:an animal model of persistent peripheral neuropathic pain[J]. Pain,2000,87(2):149-58.
[10]Ringe JD, Body JJ. A review of bone pain relief with ibandronate and other bisphosphonates in disorders of increased bone turnover[J]. Clin Exp Rheumatol,2007, 25(5):766-74.
[11]Sabino MA, Mantyh PW. Pathophysiology of bone cancer pain[J]. J Support Oncol, 2005,3(1):15-24.
[12]Luger NM, Mach DB, Sevcik MA, et al. Bone cancer pain:from model to mechanism to therapy[J]. J Pain Symptom Manage,2005,29(5 Suppl):S32-46.
[13]Kawamata T, Omote K. Activation of spinal N-methyl-D-aspartate receptors stimulates a nitric oxide/cyclic guanosine 3,5-monophosphate/glutamate release cascade in nociceptive signaling[J]. Anesthesiology,1999,91(5):1415-24.
[14]Boyce S, Wyatt A, Webb JK, et al. Selective NMDA NR2B antagonists induce antinociception without motor dysfunction:correlation with restricted localisation of NR2B subunit in dorsal horn. [J]. Neuropharmacology,1999,38(5):611-623.
[1]Porreca F, Ossipov MH, Gebhart GF. Chronic pain and medullary descending facilitation. Trends Neurosci,2002,25:319-325.
[2]Fields, H.L. and Basbaum, A.I. Central nervous system mechanisms of pain modulation. In Textbook of Pain 1999 (Wall, P.D. and Melzack R. eds), pp.309-329,Churchill Livingstone.
[3]Millan MJ. Descending control of pain. Prog Neurobiol,2002,66:355-474.
[4]Zhuo M, Gebhart GF. Modulation of noxious and non-noxious spinal mechanical transmission from the rostral medial medulla in the rat. J Neurophysiol,2002,88: 2928-2941.
[5]Neubert MJ, Kincaid W, Heinricher MM. Nociceptive facilitating neurons in the rostral ventromedial medulla. Pain,2004,110:158-165. S.E. Burgess, L.R. Gardell, M.H. Ossipov, et al.
[6]Burgess SE, Gardell LR, Ossipov MH, et al Time-dependent descending facilitation from the rostral ventromedial medulla maintains, but does not initiate, neuropathic pain. J Neurosci,2002,22:5129-5136.
[7]Fatem-Zohra Laalou, Anne Pereira de Vasconcelos, Philippe Oberling et al. Involvement of the Basal Cholinergic Forebrain in the Mediation of General (Propofol) Anesthesia. Anesthesiology 2008; 108:888-96
[8]D.V. Tillu, G.F. Gebhart, K.A. Sluka. Descending facilitatory pathways from the RVM initiate and maintain bilateral hyperalgesia after muscle insult[J]. Pain,2008,136(3):331-9.
[9]Porreca F, Burgess SE, Gardell LR, et al. Inhibition of neuropathic pain by selective ablation of brainstem medullary cells expressing the mu-opioid receptor. J Neurosci,2001, 21(14):5281-5288.
[10]Vera-Portocarrero LP, Zhang ET, Ossipov MH, et al. Descending facilitation from the rostral ventromedial medulla maintains nerve injury-induced central sensitization. Neuroscience,2006,140(4):1311-1320.
[11]Dixon WJ. Efficient analysis of experimental observations. Annu Rev Pharmacol Toxicol,1980,20:441-462.
[12]Chaplan SR, Bach FW, Pogrel JW, et al. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods,1994,53(1):55-63.
[13]Decosterd I, Woolf CJ. Spared nerve injury:an animal model of persistent peripheral neuropathic pain. Pain,2000,87(2):149-58.
[14]Urch CE, Donovan-Rodriguez T, Gordon-Williams R, et al. Efficacy of chronic morphine in a rat model of cancer-induced bone pain:behavior and in dorsal horn pathophysiology. J Pain,2005,6(12):837-845.
[15]F. Wei, R. Dubner, K. Ren, Nucleus reticularis gigantocellularis and nucleus raphe magnus in the brain stem exert opposite effects on behavioral hyperalgesia and spinal Fos protein expression after peripheral inflammation, Pain 80 (1999) 127-141.
[16]Horacio Vanegas. To the descending pain-control system in rats, inflammation-induced primary and secondary hyperalgesia are two different things. Neuroscience Letters 361 2004,361:225-228
[17]Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous System D Goula, JS Remy, P Erbacher, M Wasowicz, et al. Gene Therapy (1998) 5,712-717
[18]Behr JP. Gene transfer with synthetic cationic amphiphiles:prospects for gene therapy. Bioconj Chem.1994,5:382-389.
[19]Boussif O et al. A novel, versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo:polyethylenimine. Proc Natl Acad Sci USA.1995,92: 7297-7303.
[20]Boussif O, Zanta MA, Behr JP. Optimized galenics improve in vitro gene transfer with cationic molecules up to a thousandfold. Gene Therapy.1996,3:1074-1080.
[21]Schwartz B et al. Lipospermine-based gene transfer into the newborn mouse brain is optimized by a low lipospermine/DNA charge ratio. Hum Gene Ther 1995; 6:1515-1524.
[22]Abdallah B, Sachs L, Demeneix BA. Non-viral gene transfer:applications in developmental biology and gene transfer. Biol Cell.1995,85:1-7.
[23]Abdallah B et al. A powerful non-viral vector for in vivo gene transfer into the adult mammalian brain:polyethylenimine. Hum Gene Ther 1996; 7:1947-1957.
[24]Behr, J. P. and Demeneix, B. A. Gene delivery with polycationic amphiphiles and polymers. Curr. Res. Mol. Ther.1998,1:5-12.
[1]C.E. Thomas, A. Ehrhardt, M.A. Kay, Progress and problems with the use of viral vectors for gene therapy, Nat. Rev.,Genet.2003,4:346-358.
[2]T. Hollon, Researchers and regulators reflect on first gene therapy death, Nat. Med. 2000,6
[3]Assessment of adenoviral vector safety and toxicity:report of the National Institutes of Health Recombinant DNA Advisory Committee, Hum. Gene Ther.2002,13:3-13.
[4]E. Marshall, Gene therapy death prompts review of adenovirus vector, Science.1999, 286:2244-2245.
[5]G.D. Schmidt-Wolf, I.G.H. Schmidt-Wolf, Non-viral and hybrid vectors in human gene therapy:an update, Trends Mol. Med.2003,9:67-72.
[6]A. Kichler, Gene transfer with modified polyethylenimines, J. Gene Med.2004,6: S3-S10.
[7]M. Neu, D. Fischer, T. Kissel, Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives, J. Gene Med.2005,7:992-1009.
[8]T.G. Park, J.H. Jeong, S.W.Kim. Current status of polymeric gene delivery systems, Adv. Drug Deliv. Rev.2006,58:467-486.
[9]S. Zhang, Y. Xu, B. Wang, W. Qiao, D. Liu, Z. Li, Cationic compounds used in lipoplexes and polyplexes for gene delivery, J. Control. Release.2004,100:165-180.
[10]M. Lee, S.W. Kim, Polyethylene glycol-conjugated copolymers for plasmid DNA delivery, Pharm. Res.2005,22:1-10.
[11]T. Bronich, A. Kabanov, L.A. Marky, A thermodynamic characterization of the interaction of a cationic polymer with DNA, J. Phys. Chem.,2001, B 105:6041-6050.
[12]M.X. Tang, F.C. Szoka Jr. The influence of polymer structure on the interaction of cationic polymers with DNA and morphology of the resulting complexes. Gene Ther. 1997,4:823-832.
[13]Y.K. Oh, D. Suh, J.M. Kim, H.G. Choi, K. Shin, J.J. Ko, Polyethylenimine-mediated cellular uptake, nucleus trafficking and expression of cytokine plasmid DNA, Gene Ther. 2002,9:1627-1632.
[14]V.A. Broomfield, DNA condensation, Curr. Opin. Struck, Biol.1996,6:334-341.
[15]O. Boussif, H.F. Lezoualc, A.M. Zanta, et al. Aversatile vector for gene and oligonucleotide transfer into cells in culture and in vivo:polyethylenimine, Proc. Natl. Acad. Sci. U. S. A.1995,92:7297-7301.
[16]W.T. Godbey, K.K.Wu, A.G. Mikos, Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery, Proc. Natl. Acad. Sci. U. S. A.1999, 96:5177-5181.
[17]J.P. Clamme, G. Krishnamoorthy, Y. Mely, Intracellular dynamics of the gene delivery vehicle polyethylenimine during transfection:investigation by two-photon fluorescence correlation spectroscopy, Biochim. Biophys. Acta, Biomembr.2003,1617:52-61.
[18]M.L. Forrest, D.W. Pack, On the kinetics of polyplex endocytic trafficking: implications for gene delivery vector design, Molec. Ther.2002,6:57-66.
[19]Abdallah, B., Hassan, A., Benoist, C., et al. Powerful nonviral vector for in vivo gene transfer into the adult mammalian brain:polyethylenimine. Hum. Gene Ther.1996,7: 1947-1954.
[20]Martres, M. P., Demeneix, B. A., Hanoun, N et al. Up-and down-expression of dopamine transporter by plasmid DNA transfer in the rat brain. Eur. J. Neurosc.1998:10, 3607-3616.
[21]Goula, D., Remy, J., Erbacher, P.et al. Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system. Gene Ther.1998a,5: 712-717.
[22]Brunner, S., Furtbauer, E., Sauer, T et al. Overcoming the nuclear barrier:cell cycle independent nonviral gene transfer with linear polyethylenimine or electroporation. Mol Ther 2002,5:80-6.
[23]Bragonzi, A. et al. Comparison between cationic polymers and lipids in mediating systemic gene delivery to the lungs. Gene Ther 1999:6,1995-2004
[24]Chemin, I. et al. Liver-directed gene transfer:a linear polyethlenimine derivative mediates highly efficient DNA delivery to primary hepatocytes in vitro and in vivo. J Viral Hepat 1998,5:369-75.
[25]Ferrari, S. et al. Polyethylenimine shows properties of interest for cystic fibrosis gene therapy. Biochim Biophys Acta.1999,1447:219-25.
[26]Guissouma, H. et al. Feedback on hypothalamic TRH transcription is dependent on thyroid hormone receptor N terminus. Mol Endocrinol.2002,16:1652-66.
[27]Lemkine, G. F. et al. Preferential transfection of adult mouse neural stem cells and their immediate progeny in vivo with polyethylenimine. Mol Cell Neurosci.2002,19: 165-74.
[28]Wu, K. et al. Polyethylenimine-mediated NGF gene delivery protects transected septal cholinergic neurons. Brain Res,2004,1008:284-7.
[29]Meyerson BA. Neurosurgical approaches to pain treatment. Acta Anaesthesiol Scand 2001,45(9):1108-1113.
[30]Slavik E, Ivanovic S. Cancer pain (neurosurgical management). Acta Chir Iugosl 2004, 51(4):15-23.
[31]Kanpolat Y. Percutaneous destructive pain procedures on the upper spinal cord and
brain stem in cancer pain:CT-guided techniques, indications and results. Adv Tech Stand Neurosurg 2007,32:147-173.
[32]Guastella V, Mick G, Laurent B. Non pharmacologic treatment of neuropathic pain. Presse Med.2008,37(2 Pt 2):354-357.
[33]Mason P. Contributions of the medullary raphe and ventromedial reticular region to pain modulation and other homeostatic functions. Annual review of neuroscience 2001, 24:737-777.
[34]Mason P. Ventromedial medulla:pain modulation and beyond. J Comp Neurol 2005b, 493(1):2-8.
[1]Porreca F, Ossipov MH, Gebhart GF. Chronic pain and medullary descending facilitation. Trends Neurosci,2002,25:319-325.
[2]Fields, H.L. and Basbaum, A.I. Central nervous system mechanisms of pain modulation. In Textbook of Pain 1999 (Wall, P.D. and Melzack R. eds), pp.309-329,Churchill Livingstone.
[3]Millan MJ. Descending control of pain. Prog Neurobiol,2002,66:355-474.
[4]Vanegas H, Schaible HG Descending control of persistent pain:inhibitory or facilitatory? Brain Res Brain Res Rev,2004,46:295-309.
[5]Basbaum AI, Fields HL. Endogenous pain control systems:brainstem spinal pathways and endorphin circuitry. Annu Rev Neurosci,1984,7:309-338.
[6]Xu M, Kim CJ, Neubert MJ, Heinricher MM.et al. NMDA receptor-mediated activation of medullary pro-nociceptive neurons is required for secondary thermal hyperalgesia. Pain, 2007,127:253-262.
[7]Evidence for an intrinsic mechanism of antinoceptive tolerance within the ventrolateral periaqueductal gray of rats. Lane DA, Patel PA, PATEL, Morgan MM. Neuroscience 135 (2005)227-234
[8]Zhuo M, Gebhart GF. Modulation of noxious and non-noxious spinal mechanical transmission from the rostral medial medulla in the rat. J Neurophysiol,2002,88: 2928-2941.
[9]Mason P. Ventromedial medulla:pain modulation and beyond. J Comp Neurol.2005, 493:2-8.
[10]Soper WY, Melzack R. Stimulation-produced analgesia:evidence for somatotopic organization in the midbrain. Brain Res,1982,251:301-31
[11]Watkins LR, Griffin G, Leichnetz GR, Mayer DJ. The somatotopic organization of the nucleus raphe magnus and surrounding brain stem structures as revealed by HRP slow-release gels. Brain Res 1980,181:1-15
[12]Neubert MJ, Kincaid W, Heinricher MM. Nociceptive facilitating neurons in the rostral ventromedial medulla. Pain 2004; 110:158-65.
[13]Jens Ellrich, Coskun Ulucan,Cathrin Schnell.Are 'neutral cells' in the rostral ventro-medial medulla subtypes of on-and off-cells? Neuroscience Research.2000,38: 419-423
[14]Heinricher MM, Neubert MJ. Neural basis for the hyperalgesic action of cholecystokinin in the rostral ventromedial medulla. J Neurophysiol 2004;92:1982-9.
[15]Z.Z. Pan, J.T. Williams, P.B. Osborne, Opioid actions on single nucleus raphe magnus neurons from rat and guinea-pig in vitro, J. Physiol. (Lond.) 1990,427:519-532
[16]Kincaid W, Neubert MJ, Xu M, Kim CJ, Heinricher MM. Role for medullary pain facilitating neurons in secondary thermal hyperalgesia. J Neurophysiol 2006; 95:33-41.
[17]Martin G, Montagne CJ, Oliveras JL. Involvement of ventromedial medulla "multimodal, multireceptive" neurons in opiate spinal descending control system:a single-unit study of the effect of morphine in the awake, freely moving rat. J Neurosci 1992, 12:1511-1522.
[18]Mason P, Escobedo I, Burgin C, Bergan J, Lee JH, Last EJ, Holub A. Nociceptive responsiveness during slow wave sleep and waking in the rat. Sleep 2001,24:32-38
[19]Foo H, Mason P. Bicuculline microinjection into the raphe magnus suppresses air puff-evoked awakenings in behaving rats. Soc Neurosci Abstr,2000,26:660.
[20]Jens Ellrich · Coskun Ulucan · Cathrin Schnell. Is the response pattern of on-and off-cells in the rostral ventromedial medulla to noxious stimulation independent of stimulation site? Exp Brain Res,2001,136:394-399
[21]Horacio Vanegas. To the descending pain-control system in rats, inflammation-induced primary and secondary hyperalgesia are two different things. Neuroscience Letters,2004, 361:225-228
[22]K. Ren, R. Dubner, Enhanced descending modulation of nociception in rats with persistent hindpaw inflammation, J. Neurophysiol.1996,76:3025-3037.
[23]. M. Tsuruoka, W.D. Willis, Bilateral lesions in the area of the nucleus locus coeruleus affect the development of hyperalgesia during carrageenan-induced inflammation, Brain Res.1996,726:233-236.
[24]R.W. Hurley, D.L. Hammond, The analgesic effects of supraspinal μand δopioid receptor agonists are potentiated during persistent inflammation, J. Neurosci.2000,20: 1249-1259.
[25]R.W. Hurley, D.L. Hammond, Contribution of endogenous enkephalins to the enhanced analgesic effects of supraspinal A opioid receptor agonists after inflammatory injury, J. Neurosci.2001,21:2536-2545.
[26]Y. Guan, W. Guo, S.-P. Zou, R. Dubner, K. Ren, Inflammationinduced upregulation of AMPA receptor subunit expression in brain stem pain modulatory circuitry, Pain,2003,104: 401-413.
[27]Y. Guan, R. Terayama, R. Dubner, K. Ren, Plasticity in excitatory amino acid receptor-mediated descending pain modulation after inflammation. J. Pharmacol. Exp. Ther. 2002,300:513-520.
[28]K. Miki, Q.Q. Zhou, W. Guo, Y. Guan, R. Terayama, R. Dubner, K. Ren, Change in gene expression and neuronal phenotype in brain stem pain modulatory circuitry after inflammation, J. Neurophysiol.2002,87:750-760.
[29]R. Terayama, R. Dubner, K. Ren, The roles of NMD A receptor activation and nucleus reticularis gigantocellularis in the time dependent changes in descending inhibition after inflammation, Pain,2002,97:171-181.
[30]M.O. Urban, S.V. Coutinho, GF. Gebhart, Involvement of excitatory amino acid receptors and nitric oxide in the rostral ventromedial medulla in modulating secondary hyperalgesia produced by mustard oil, Pain,1999,81:45-55.
[31]S.E. Burgess, L.R. Gardell, M.H. Ossipov, T.P. Malan, T.W. Vanderah, J. Lai, F. Porreca, Time-dependent descending facilitation from the rostral ventromedial medulla maintains, but does not initiate, neuropathic pain, J. Neurosci.2002,22:5129-5136.
[32]M.M. Heinricher, S. McGaraughty, V. Tortorici, Circuitry underlying anti-opioid actions of cholecystokinin within the rostral ventromedial medulla, J. Neurophysiol.2001 85:280-286.
[33]C.J. Kovelowski, M.H. Ossipov, H. Sun, J. Lai, T.P. Malan, F. Porreca, Supraspinal cholecystokinin may drive tonic descending facilitation mechanisms to maintain neuropathic pain in the rat, Pain,2000,87:265-273.
[34]F. Porreca, S.E. Burgess, L.R. Gardell, T.W. Vanderah, T.P. Malan, M.H. Ossipov, D.A. Lappi, J. Lai, Inhibition of neuropathic pain by selective ablation of brainstem medullary cells expressing the A-opioid receptor, J. Neurosci,2001,21:5281-5288.
[35]L.R. Gardell, T.W. Vanderah, S.E. Gardell, R. Wang, M.H. Osipov, J. Lai, F. Porreca, Enhanced evoked neurotransmitter release in experimental neuropathy requires descending facilitation, J. Neurosci.2003,23:8370-8379.
[36]D. Bian, M.H. Ossipov, C. Zhong, T.P. Malan, F. Porreca, Tactile allodynia, but not thermal hyperalgesia, of the hindlimb is blocked by spinal transection in rats with nerve injury, Neurosci. Lett,1998,241:79-82.
[37]R. Monhemius, D.L. Green, M.H.T. Roberts, J. Azami, Periaqueductal grey mediated inhibition of responses to noxious stimulation is dynamically activated in a rat model of neuropathic pain, Neurosci. Lett.2001,298:70-74.
2. Savill J. Apoptosis in resolution of inflammation. J Leuk Biol 1997,61:375-380.
3. Ting-Jun FAN, Li-Hui HAN, Ri-Shan CONG et al. Caspase Family Proteases and Apoptosis. Acta Biochimica et Biophysica Sinica 2005,37(11):719-727.
4. Shi Y. Caspase activation, inhibition, and reactivation:a mechanistic view. Protein Sci, 2004,13(8):1979-1987.
5. Fadeel B and Orrenius S. Apoptosis:a basic biological phenomenon with wide-ranging implications in human disease. J Intern Med,2005,258(6):479-517.
6. Hengartner MO. The biochemistry of apoptosis. Nature 2000,407(6805):770-776.
7. Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol,2004,5(11):897-907.
8. Srinivasula SM, Ahmad M, MacFarlane M, et al. Generation of constitutively active recombinant caspases-3 and-6 by rearrangement of their subunits. J Biol Chem,1998, 273(17):10107-10111.
9. Fischer U and Schulze-Osthoff K. Apoptosis-based therapies and drug targets. Cell Death Differ,2005,12 Suppl 1:942-961.
10. Chelur DS and Chalfie M. Targeted cell killing by reconstituted caspases. Proc Natl Acad Sci U S A,2007,104(7):2283-2288.
11. Jia LT, Zhang LH, Yu CJ, et al. Specific tumoricidal activity of a secreted proapoptotic protein consisting of HER2 antibody and constitutively active caspase-3. Cancer Res,2003, 63(12):3257-3262.
12. Chelur DS and Chalfie M. Targeted cell killing by reconstituted caspases. Proc Natl Acad Sci U S A,2007,104(7):2283-2288.
13.高峰,田玉科,杨辉等.猿肾病毒SV40大T抗原基因永生化大鼠神经前体细胞株的构建.中华麻醉学杂志,2005,25：597-600.
14. Hengartner MO. The biochemistry of apoptosis. Nature,2000,407(6805):770-776
[1]Slavin KV, Tesoro EP, Mucksavage JJ. The treatment of cancer pain. Drugs Today (Barc) 2004;40:235-45. Biology of Bone Cancer Pain Michael J. Goblirsch, Pawel P. Zwolak, and Denis R. Clohisy Clin Cancer Res 2006;12(20 Suppl) October 15,2006.
[2]Bone Cancer Pain:From Model to Mechanism to Therapy. Nancy M. Luger, BA, David B. Mach, BS, Molly A. Journal of Pain and Symptom Management Vol.29 No.5S May 2005
[3]Cherny NI. The management of cancer pain. Ca Cancer J Clin 2000;50:70-116. quiz 117-120.
[4]Koshy RC, Grossman SA. Treatment of bone pain. In:Body JJ, ed. Tumor bone diseases and osteoporosis in cancer patients. New York:Marcel Dekker, Inc,2000:245-261.
[5]Medhurst SJ, Walker K, Bowes M, et al. A rat model of bone cancer pain. Pain,2002, 96(1-2):129-140.
[6]Mao-Ying QL, Zhao J, Dong ZQ et al. A rat model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells[J]. Biochem Biophys Res Commun,2006,345(4):1292-1298.
[7]Walker 256 Tumor-Bearing Rats as a Model to Study Cancer Pain. Patricia Brigatte, Sandra Coccuzzo Sampaio, Vanessa Pacciari Gutierrez et al. The Journal of Pain, Vol 8, No 5 (May),2007:pp 412-421
[8]Chaplan SR, Bach FW, Pogrel JW et al. Quantitative assessment of tactile allodynia in the rat paw[J]. J Neurosci Methods,1994,53(1):55-63.
[9]Decosterd I, Woolf CJ. Spared nerve injury:an animal model of persistent peripheral neuropathic pain[J]. Pain,2000,87(2):149-58.
[10]Ringe JD, Body JJ. A review of bone pain relief with ibandronate and other bisphosphonates in disorders of increased bone turnover[J]. Clin Exp Rheumatol,2007, 25(5):766-74.
[11]Sabino MA, Mantyh PW. Pathophysiology of bone cancer pain[J]. J Support Oncol, 2005,3(1):15-24.
[12]Luger NM, Mach DB, Sevcik MA, et al. Bone cancer pain:from model to mechanism to therapy[J]. J Pain Symptom Manage,2005,29(5 Suppl):S32-46.
[13]Kawamata T, Omote K. Activation of spinal N-methyl-D-aspartate receptors stimulates a nitric oxide/cyclic guanosine 3,5-monophosphate/glutamate release cascade in nociceptive signaling[J]. Anesthesiology,1999,91(5):1415-24.
[14]Boyce S, Wyatt A, Webb JK, et al. Selective NMDA NR2B antagonists induce antinociception without motor dysfunction:correlation with restricted localisation of NR2B subunit in dorsal horn. [J]. Neuropharmacology,1999,38(5):611-623.
[1]Porreca F, Ossipov MH, Gebhart GF. Chronic pain and medullary descending facilitation. Trends Neurosci,2002,25:319-325.
[2]Fields, H.L. and Basbaum, A.I. Central nervous system mechanisms of pain modulation. In Textbook of Pain 1999 (Wall, P.D. and Melzack R. eds), pp.309-329,Churchill Livingstone.
[3]Millan MJ. Descending control of pain. Prog Neurobiol,2002,66:355-474.
[4]Zhuo M, Gebhart GF. Modulation of noxious and non-noxious spinal mechanical transmission from the rostral medial medulla in the rat. J Neurophysiol,2002,88: 2928-2941.
[5]Neubert MJ, Kincaid W, Heinricher MM. Nociceptive facilitating neurons in the rostral ventromedial medulla. Pain,2004,110:158-165. S.E. Burgess, L.R. Gardell, M.H. Ossipov, et al.
[6]Burgess SE, Gardell LR, Ossipov MH, et al Time-dependent descending facilitation from the rostral ventromedial medulla maintains, but does not initiate, neuropathic pain. J Neurosci,2002,22:5129-5136.
[7]Fatem-Zohra Laalou, Anne Pereira de Vasconcelos, Philippe Oberling et al. Involvement of the Basal Cholinergic Forebrain in the Mediation of General (Propofol) Anesthesia. Anesthesiology 2008; 108:888-96
[8]D.V. Tillu, G.F. Gebhart, K.A. Sluka. Descending facilitatory pathways from the RVM initiate and maintain bilateral hyperalgesia after muscle insult[J]. Pain,2008,136(3):331-9.
[9]Porreca F, Burgess SE, Gardell LR, et al. Inhibition of neuropathic pain by selective ablation of brainstem medullary cells expressing the mu-opioid receptor. J Neurosci,2001, 21(14):5281-5288.
[10]Vera-Portocarrero LP, Zhang ET, Ossipov MH, et al. Descending facilitation from the rostral ventromedial medulla maintains nerve injury-induced central sensitization. Neuroscience,2006,140(4):1311-1320.
[11]Dixon WJ. Efficient analysis of experimental observations. Annu Rev Pharmacol Toxicol,1980,20:441-462.
[12]Chaplan SR, Bach FW, Pogrel JW, et al. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods,1994,53(1):55-63.
[13]Decosterd I, Woolf CJ. Spared nerve injury:an animal model of persistent peripheral neuropathic pain. Pain,2000,87(2):149-58.
[14]Urch CE, Donovan-Rodriguez T, Gordon-Williams R, et al. Efficacy of chronic morphine in a rat model of cancer-induced bone pain:behavior and in dorsal horn pathophysiology. J Pain,2005,6(12):837-845.
[15]F. Wei, R. Dubner, K. Ren, Nucleus reticularis gigantocellularis and nucleus raphe magnus in the brain stem exert opposite effects on behavioral hyperalgesia and spinal Fos protein expression after peripheral inflammation, Pain 80 (1999) 127-141.
[16]Horacio Vanegas. To the descending pain-control system in rats, inflammation-induced primary and secondary hyperalgesia are two different things. Neuroscience Letters 361 2004,361:225-228
[17]Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous System D Goula, JS Remy, P Erbacher, M Wasowicz, et al. Gene Therapy (1998) 5,712-717
[18]Behr JP. Gene transfer with synthetic cationic amphiphiles:prospects for gene therapy. Bioconj Chem.1994,5:382-389.
[19]Boussif O et al. A novel, versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo:polyethylenimine. Proc Natl Acad Sci USA.1995,92: 7297-7303.
[20]Boussif O, Zanta MA, Behr JP. Optimized galenics improve in vitro gene transfer with cationic molecules up to a thousandfold. Gene Therapy.1996,3:1074-1080.
[21]Schwartz B et al. Lipospermine-based gene transfer into the newborn mouse brain is optimized by a low lipospermine/DNA charge ratio. Hum Gene Ther 1995; 6:1515-1524.
[22]Abdallah B, Sachs L, Demeneix BA. Non-viral gene transfer:applications in developmental biology and gene transfer. Biol Cell.1995,85:1-7.
[23]Abdallah B et al. A powerful non-viral vector for in vivo gene transfer into the adult mammalian brain:polyethylenimine. Hum Gene Ther 1996; 7:1947-1957.
[24]Behr, J. P. and Demeneix, B. A. Gene delivery with polycationic amphiphiles and polymers. Curr. Res. Mol. Ther.1998,1:5-12.
[1]C.E. Thomas, A. Ehrhardt, M.A. Kay, Progress and problems with the use of viral vectors for gene therapy, Nat. Rev.,Genet.2003,4:346-358.
[2]T. Hollon, Researchers and regulators reflect on first gene therapy death, Nat. Med. 2000,6
[3]Assessment of adenoviral vector safety and toxicity:report of the National Institutes of Health Recombinant DNA Advisory Committee, Hum. Gene Ther.2002,13:3-13.
[4]E. Marshall, Gene therapy death prompts review of adenovirus vector, Science.1999, 286:2244-2245.
[5]G.D. Schmidt-Wolf, I.G.H. Schmidt-Wolf, Non-viral and hybrid vectors in human gene therapy:an update, Trends Mol. Med.2003,9:67-72.
[6]A. Kichler, Gene transfer with modified polyethylenimines, J. Gene Med.2004,6: S3-S10.
[7]M. Neu, D. Fischer, T. Kissel, Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives, J. Gene Med.2005,7:992-1009.
[8]T.G. Park, J.H. Jeong, S.W.Kim. Current status of polymeric gene delivery systems, Adv. Drug Deliv. Rev.2006,58:467-486.
[9]S. Zhang, Y. Xu, B. Wang, W. Qiao, D. Liu, Z. Li, Cationic compounds used in lipoplexes and polyplexes for gene delivery, J. Control. Release.2004,100:165-180.
[10]M. Lee, S.W. Kim, Polyethylene glycol-conjugated copolymers for plasmid DNA delivery, Pharm. Res.2005,22:1-10.
[11]T. Bronich, A. Kabanov, L.A. Marky, A thermodynamic characterization of the interaction of a cationic polymer with DNA, J. Phys. Chem.,2001, B 105:6041-6050.
[12]M.X. Tang, F.C. Szoka Jr. The influence of polymer structure on the interaction of cationic polymers with DNA and morphology of the resulting complexes. Gene Ther. 1997,4:823-832.
[13]Y.K. Oh, D. Suh, J.M. Kim, H.G. Choi, K. Shin, J.J. Ko, Polyethylenimine-mediated cellular uptake, nucleus trafficking and expression of cytokine plasmid DNA, Gene Ther. 2002,9:1627-1632.
[14]V.A. Broomfield, DNA condensation, Curr. Opin. Struck, Biol.1996,6:334-341.
[15]O. Boussif, H.F. Lezoualc, A.M. Zanta, et al. Aversatile vector for gene and oligonucleotide transfer into cells in culture and in vivo:polyethylenimine, Proc. Natl. Acad. Sci. U. S. A.1995,92:7297-7301.
[16]W.T. Godbey, K.K.Wu, A.G. Mikos, Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery, Proc. Natl. Acad. Sci. U. S. A.1999, 96:5177-5181.
[17]J.P. Clamme, G. Krishnamoorthy, Y. Mely, Intracellular dynamics of the gene delivery vehicle polyethylenimine during transfection:investigation by two-photon fluorescence correlation spectroscopy, Biochim. Biophys. Acta, Biomembr.2003,1617:52-61.
[18]M.L. Forrest, D.W. Pack, On the kinetics of polyplex endocytic trafficking: implications for gene delivery vector design, Molec. Ther.2002,6:57-66.
[19]Abdallah, B., Hassan, A., Benoist, C., et al. Powerful nonviral vector for in vivo gene transfer into the adult mammalian brain:polyethylenimine. Hum. Gene Ther.1996,7: 1947-1954.
[20]Martres, M. P., Demeneix, B. A., Hanoun, N et al. Up-and down-expression of dopamine transporter by plasmid DNA transfer in the rat brain. Eur. J. Neurosc.1998:10, 3607-3616.
[21]Goula, D., Remy, J., Erbacher, P.et al. Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system. Gene Ther.1998a,5: 712-717.
[22]Brunner, S., Furtbauer, E., Sauer, T et al. Overcoming the nuclear barrier:cell cycle independent nonviral gene transfer with linear polyethylenimine or electroporation. Mol Ther 2002,5:80-6.
[23]Bragonzi, A. et al. Comparison between cationic polymers and lipids in mediating systemic gene delivery to the lungs. Gene Ther 1999:6,1995-2004
[24]Chemin, I. et al. Liver-directed gene transfer:a linear polyethlenimine derivative mediates highly efficient DNA delivery to primary hepatocytes in vitro and in vivo. J Viral Hepat 1998,5:369-75.
[25]Ferrari, S. et al. Polyethylenimine shows properties of interest for cystic fibrosis gene therapy. Biochim Biophys Acta.1999,1447:219-25.
[26]Guissouma, H. et al. Feedback on hypothalamic TRH transcription is dependent on thyroid hormone receptor N terminus. Mol Endocrinol.2002,16:1652-66.
[27]Lemkine, G. F. et al. Preferential transfection of adult mouse neural stem cells and their immediate progeny in vivo with polyethylenimine. Mol Cell Neurosci.2002,19: 165-74.
[28]Wu, K. et al. Polyethylenimine-mediated NGF gene delivery protects transected septal cholinergic neurons. Brain Res,2004,1008:284-7.
[29]Meyerson BA. Neurosurgical approaches to pain treatment. Acta Anaesthesiol Scand 2001,45(9):1108-1113.
[30]Slavik E, Ivanovic S. Cancer pain (neurosurgical management). Acta Chir Iugosl 2004, 51(4):15-23.
[31]Kanpolat Y. Percutaneous destructive pain procedures on the upper spinal cord and
brain stem in cancer pain:CT-guided techniques, indications and results. Adv Tech Stand Neurosurg 2007,32:147-173.
[32]Guastella V, Mick G, Laurent B. Non pharmacologic treatment of neuropathic pain. Presse Med.2008,37(2 Pt 2):354-357.
[33]Mason P. Contributions of the medullary raphe and ventromedial reticular region to pain modulation and other homeostatic functions. Annual review of neuroscience 2001, 24:737-777.
[34]Mason P. Ventromedial medulla:pain modulation and beyond. J Comp Neurol 2005b, 493(1):2-8.
[1]Porreca F, Ossipov MH, Gebhart GF. Chronic pain and medullary descending facilitation. Trends Neurosci,2002,25:319-325.
[2]Fields, H.L. and Basbaum, A.I. Central nervous system mechanisms of pain modulation. In Textbook of Pain 1999 (Wall, P.D. and Melzack R. eds), pp.309-329,Churchill Livingstone.
[3]Millan MJ. Descending control of pain. Prog Neurobiol,2002,66:355-474.
[4]Vanegas H, Schaible HG Descending control of persistent pain:inhibitory or facilitatory? Brain Res Brain Res Rev,2004,46:295-309.
[5]Basbaum AI, Fields HL. Endogenous pain control systems:brainstem spinal pathways and endorphin circuitry. Annu Rev Neurosci,1984,7:309-338.
[6]Xu M, Kim CJ, Neubert MJ, Heinricher MM.et al. NMDA receptor-mediated activation of medullary pro-nociceptive neurons is required for secondary thermal hyperalgesia. Pain, 2007,127:253-262.
[7]Evidence for an intrinsic mechanism of antinoceptive tolerance within the ventrolateral periaqueductal gray of rats. Lane DA, Patel PA, PATEL, Morgan MM. Neuroscience 135 (2005)227-234
[8]Zhuo M, Gebhart GF. Modulation of noxious and non-noxious spinal mechanical transmission from the rostral medial medulla in the rat. J Neurophysiol,2002,88: 2928-2941.
[9]Mason P. Ventromedial medulla:pain modulation and beyond. J Comp Neurol.2005, 493:2-8.
[10]Soper WY, Melzack R. Stimulation-produced analgesia:evidence for somatotopic organization in the midbrain. Brain Res,1982,251:301-31
[11]Watkins LR, Griffin G, Leichnetz GR, Mayer DJ. The somatotopic organization of the nucleus raphe magnus and surrounding brain stem structures as revealed by HRP slow-release gels. Brain Res 1980,181:1-15
[12]Neubert MJ, Kincaid W, Heinricher MM. Nociceptive facilitating neurons in the rostral ventromedial medulla. Pain 2004; 110:158-65.
[13]Jens Ellrich, Coskun Ulucan,Cathrin Schnell.Are 'neutral cells' in the rostral ventro-medial medulla subtypes of on-and off-cells? Neuroscience Research.2000,38: 419-423
[14]Heinricher MM, Neubert MJ. Neural basis for the hyperalgesic action of cholecystokinin in the rostral ventromedial medulla. J Neurophysiol 2004;92:1982-9.
[15]Z.Z. Pan, J.T. Williams, P.B. Osborne, Opioid actions on single nucleus raphe magnus neurons from rat and guinea-pig in vitro, J. Physiol. (Lond.) 1990,427:519-532
[16]Kincaid W, Neubert MJ, Xu M, Kim CJ, Heinricher MM. Role for medullary pain facilitating neurons in secondary thermal hyperalgesia. J Neurophysiol 2006; 95:33-41.
[17]Martin G, Montagne CJ, Oliveras JL. Involvement of ventromedial medulla "multimodal, multireceptive" neurons in opiate spinal descending control system:a single-unit study of the effect of morphine in the awake, freely moving rat. J Neurosci 1992, 12:1511-1522.
[18]Mason P, Escobedo I, Burgin C, Bergan J, Lee JH, Last EJ, Holub A. Nociceptive responsiveness during slow wave sleep and waking in the rat. Sleep 2001,24:32-38
[19]Foo H, Mason P. Bicuculline microinjection into the raphe magnus suppresses air puff-evoked awakenings in behaving rats. Soc Neurosci Abstr,2000,26:660.
[20]Jens Ellrich · Coskun Ulucan · Cathrin Schnell. Is the response pattern of on-and off-cells in the rostral ventromedial medulla to noxious stimulation independent of stimulation site? Exp Brain Res,2001,136:394-399
[21]Horacio Vanegas. To the descending pain-control system in rats, inflammation-induced primary and secondary hyperalgesia are two different things. Neuroscience Letters,2004, 361:225-228
[22]K. Ren, R. Dubner, Enhanced descending modulation of nociception in rats with persistent hindpaw inflammation, J. Neurophysiol.1996,76:3025-3037.
[23]. M. Tsuruoka, W.D. Willis, Bilateral lesions in the area of the nucleus locus coeruleus affect the development of hyperalgesia during carrageenan-induced inflammation, Brain Res.1996,726:233-236.
[24]R.W. Hurley, D.L. Hammond, The analgesic effects of supraspinal μand δopioid receptor agonists are potentiated during persistent inflammation, J. Neurosci.2000,20: 1249-1259.
[25]R.W. Hurley, D.L. Hammond, Contribution of endogenous enkephalins to the enhanced analgesic effects of supraspinal A opioid receptor agonists after inflammatory injury, J. Neurosci.2001,21:2536-2545.
[26]Y. Guan, W. Guo, S.-P. Zou, R. Dubner, K. Ren, Inflammationinduced upregulation of AMPA receptor subunit expression in brain stem pain modulatory circuitry, Pain,2003,104: 401-413.
[27]Y. Guan, R. Terayama, R. Dubner, K. Ren, Plasticity in excitatory amino acid receptor-mediated descending pain modulation after inflammation. J. Pharmacol. Exp. Ther. 2002,300:513-520.
[28]K. Miki, Q.Q. Zhou, W. Guo, Y. Guan, R. Terayama, R. Dubner, K. Ren, Change in gene expression and neuronal phenotype in brain stem pain modulatory circuitry after inflammation, J. Neurophysiol.2002,87:750-760.
[29]R. Terayama, R. Dubner, K. Ren, The roles of NMD A receptor activation and nucleus reticularis gigantocellularis in the time dependent changes in descending inhibition after inflammation, Pain,2002,97:171-181.
[30]M.O. Urban, S.V. Coutinho, GF. Gebhart, Involvement of excitatory amino acid receptors and nitric oxide in the rostral ventromedial medulla in modulating secondary hyperalgesia produced by mustard oil, Pain,1999,81:45-55.
[31]S.E. Burgess, L.R. Gardell, M.H. Ossipov, T.P. Malan, T.W. Vanderah, J. Lai, F. Porreca, Time-dependent descending facilitation from the rostral ventromedial medulla maintains, but does not initiate, neuropathic pain, J. Neurosci.2002,22:5129-5136.
[32]M.M. Heinricher, S. McGaraughty, V. Tortorici, Circuitry underlying anti-opioid actions of cholecystokinin within the rostral ventromedial medulla, J. Neurophysiol.2001 85:280-286.
[33]C.J. Kovelowski, M.H. Ossipov, H. Sun, J. Lai, T.P. Malan, F. Porreca, Supraspinal cholecystokinin may drive tonic descending facilitation mechanisms to maintain neuropathic pain in the rat, Pain,2000,87:265-273.
[34]F. Porreca, S.E. Burgess, L.R. Gardell, T.W. Vanderah, T.P. Malan, M.H. Ossipov, D.A. Lappi, J. Lai, Inhibition of neuropathic pain by selective ablation of brainstem medullary cells expressing the A-opioid receptor, J. Neurosci,2001,21:5281-5288.
[35]L.R. Gardell, T.W. Vanderah, S.E. Gardell, R. Wang, M.H. Osipov, J. Lai, F. Porreca, Enhanced evoked neurotransmitter release in experimental neuropathy requires descending facilitation, J. Neurosci.2003,23:8370-8379.
[36]D. Bian, M.H. Ossipov, C. Zhong, T.P. Malan, F. Porreca, Tactile allodynia, but not thermal hyperalgesia, of the hindlimb is blocked by spinal transection in rats with nerve injury, Neurosci. Lett,1998,241:79-82.
[37]R. Monhemius, D.L. Green, M.H.T. Roberts, J. Azami, Periaqueductal grey mediated inhibition of responses to noxious stimulation is dynamically activated in a rat model of neuropathic pain, Neurosci. Lett.2001,298:70-74.