PD模型大鼠神经细胞程序性凋亡通路研究及坏死性凋亡初探

发布时间：2018-08-25 20:09

【摘要】：帕金森病(Parkinson s disease，PD)是由于黑质多巴胺（Dopamine，DA）神经元进行性丢失导致纹状体DA递质减少造成的中枢神经系统退变性疾病。目前经典的细胞死亡理论，都难以确切解释PD脑组织中这种极其缓慢的神经元丢失和大量具特征性病理改变的细胞胞体残存的现象。目的：探讨PD模型大鼠黑质、纹状体中神经细胞死亡情况（程序性凋亡及坏死性凋亡），程序性凋亡通路上关键蛋白的表达及意义；研究半胱天冬氨酸蛋白酶（Cysteinyl aspartate specific proteinase，Caspase）抑制剂（Z-VAD-fmk）对6-羟基多巴胺（6-hydroxydopamine,6-OHDA）毁损PD模型大鼠的神经保护作用、对神经细胞死亡方式的影响，为探究PD发病过程中DA神经元死亡确切机制、寻找PD有效治疗方法具有实际意义。方法：（1）采用立体定位于前脑内侧束（Medial forebrain bundle，MFB）注射6-OHDA的方法建立大鼠PD模型，行为学筛选出成功PD模型大鼠，应用HE染色法和Nissl染色法对PD模型大鼠神经元进行病理学和形态学观察。（2）免疫组织化学标记检测PD大鼠凋亡通路中TNF-α、P53、P21、Bcl-2/Bax、Casepase-3的表达变化；（3）改良大鼠大脑置管方法，在大鼠大脑的MFB部位埋植套管，通过套管给予6-OHDA造模PD模型大鼠。实验动物分组：空白对照组(生理盐水)，PD模型组(6-OHDA+5%DMSO)，药物干预组(6-OHDA+Z-VAD-fmk)；行为学筛选出成功PD模型大鼠，药物干预组将Caspase抑制剂Z-VAD-fmk通过套管注射入PD大鼠MFB中，每天一次。对照组及PD模型组每天给予等量5%DMSO（Z-VAD-fmk溶剂）；（4）形态学检测：Nissl染色法观察PD模型大鼠注射Z-VAD-fmk后神经元丢失情况，电镜观察各种大鼠神经细胞超微机构的变化；（5）Real-time PCR检测各组大鼠Bcl-2/Bax、RIP1、RIP3mRNA的表达变化。结果：(1)注射6-OHDA2周后PD大鼠出现明显的典型旋转行为，Nissl染色及TH免疫组织化学染色标记显示PD模型大鼠毁损侧黑质致密部神经元数量锐减（P0.01）；(2)与对照组相比，PD模型组黑质及纹状体中TNF-α、P53、P21、Bax、Casepase-3表达上调，，具有显著差异（P0.05或P0.01），Bcl-2表达下调（P0.01）；(3)注射Caspase抑制剂Z-VAD-fmk后，PD大鼠模型动物行为学得到改善，黑质神经元丢失得到缓解，同时Bcl-2/Bax mRNA的表达上调；（4）电镜结果显示，Z-VAD-fmk在一定程度上能改善6-OHDA毁损所导致的线粒体减少、肿胀等超微结构上的变化，但部分胶质细胞出现水肿现象；（5）与模型组相比，药物干预组黑质RIP1，RIP3mRNA表达上升（P0.05），提示Z-VAD-fmk可能通过促进活化的胶质细胞坏死性凋亡（Necroptosis）而保护神经元。结论：（1）PD大鼠模型黑质及纹状体神经细胞以程序性凋亡为主；（2）给予Z-VAD-fmk后，PD大鼠模型黑质神经细胞程序性凋亡在一定程度上被抑制，出现了胶质细胞坏死性凋亡；（3）Z-VAD-fmk可能通过促进活化的胶质细胞坏死性凋亡而保护神经元。
[Abstract]:Parkinson's disease (Parkinson s disease,PD) is a degenerative disease of the central nervous system caused by progressive loss of substantia nigra dopamine (Dopamine,DA) neurons resulting in reduced DA transmitters in the striatum. At present, the classical theory of cell death is difficult to explain the phenomenon of extremely slow neuronal loss and a large number of characteristic pathological changes in PD brain tissue. Objective: to investigate the expression and significance of neuronal death (programmed apoptosis and necrotic apoptosis) in substantia nigra and striatum of PD rats and the expression of key proteins in programmed apoptosis pathway. To study the neuroprotective effect of cysteine aspartate protease (Cysteinyl aspartate specific proteinase,Caspase inhibitor (Z-VAD-fmk) on the neuroprotective effect of 6-hydroxydopamine (6-hydroxydopamine 6-OHDA) on the neuronal death in PD model rats, and to explore the exact mechanism of DA neuron death in the pathogenesis of PD. It is of practical significance to find an effective treatment for PD. Methods: (1) the rat model of PD was established by injecting 6-OHDA into the medial forebrain tract (Medial forebrain bundle,MFB). The successful PD model rats were screened out by behavioral science. HE staining and Nissl staining were used to observe the histopathology and morphology of neurons in PD model rats. (2) immunohistochemical staining was used to detect the expression of TNF- 伪 -P53C21 / BaxCasepase-3 in the apoptosis pathway of PD rats. A cannula was implanted at the MFB site of the brain of the rat, and the model PD model rats were established by using the 6-OHDA cannula. The experimental animals were divided into two groups: blank control group (normal saline) PD model group (6-OHDA 5%DMSO), drug intervention group (6-OHDA Z-VAD-fmk); successful PD model rats were screened by behavioral science. Caspase inhibitor Z-VAD-fmk was injected into MFB of PD rats once a day in the drug intervention group. The control group and PD model group were given the same amount of 5%DMSO (Z-VAD-fmk solvent); (4) every day to observe the neuronal loss after Z-VAD-fmk injection in PD model rats, and the ultrastructure changes of various nerve cells were observed by electron microscope. (5) the expression of Bcl-2/Bax,RIP1,RIP3mRNA was detected by Real-time PCR. Results: (1) the typical rotational behavior of PD rats and TH immunohistochemical staining showed that the number of neurons in the substantia nigra of PD model rats was significantly decreased (P0.01). (2) compared with the control group, the expression of TNF- 伪 P53OP21Baxepase-3 in substantia nigra and striatum in PD model group was up-regulated (P0.05 or P0.01), and the expression of Bcl-2 was down-regulated (P0.01); (3). After the injection of Caspase inhibitor Z-VAD-fmk, the behavior of PD rat model was improved, and the loss of neurons in substantia nigra was alleviated. At the same time, the expression of Bcl-2/Bax mRNA was up-regulated. (4) the results of electron microscope showed that Z-VAD-fmk could improve the ultrastructural changes of mitochondria and swelling caused by 6-OHDA damage to some extent, but some glial cells showed edema. (5) compared with the model group, ZVAD-fmk could improve the changes of mitochondria and swelling. The expression of RIP1,RIP3mRNA in substantia nigra was increased in drug intervention group (P0.05), suggesting that Z-VAD-fmk may protect neurons by promoting apoptosis of activated glial cells. Conclusion: (1) programmed apoptosis of substantia nigra and striatum neurons in PD rat model was dominant; (2) programmed apoptosis of substantia nigra neurons in PD rat model was inhibited to some extent and glial necrosis apoptosis appeared after Z-VAD-fmk administration. (3) Z-VAD-fmk may protect neurons by promoting apoptosis of activated glial cells.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5

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