【摘要】：帕金森病(Parkinson s disease，PD)是由于黑質(zhì)多巴胺（Dopamine，DA）神經(jīng)元進(jìn)行性丟失導(dǎo)致紋狀體DA遞質(zhì)減少造成的中樞神經(jīng)系統(tǒng)退變性疾病。目前經(jīng)典的細(xì)胞死亡理論，都難以確切解釋PD腦組織中這種極其緩慢的神經(jīng)元丟失和大量具特征性病理改變的細(xì)胞胞體殘存的現(xiàn)象。 目的：探討PD模型大鼠黑質(zhì)、紋狀體中神經(jīng)細(xì)胞死亡情況（程序性凋亡及壞死性凋亡），程序性凋亡通路上關(guān)鍵蛋白的表達(dá)及意義；研究半胱天冬氨酸蛋白酶（Cysteinyl aspartate specific proteinase，Caspase）抑制劑（Z-VAD-fmk）對6-羥基多巴胺（6-hydroxydopamine,6-OHDA）毀損PD模型大鼠的神經(jīng)保護(hù)作用、對神經(jīng)細(xì)胞死亡方式的影響，為探究PD發(fā)病過程中DA神經(jīng)元死亡確切機(jī)制、尋找PD有效治療方法具有實際意義。 方法：（1）采用立體定位于前腦內(nèi)側(cè)束（Medial forebrain bundle，MFB）注射6-OHDA的方法建立大鼠PD模型，行為學(xué)篩選出成功PD模型大鼠，應(yīng)用HE染色法和Nissl染色法對PD模型大鼠神經(jīng)元進(jìn)行病理學(xué)和形態(tài)學(xué)觀察。（2）免疫組織化學(xué)標(biāo)記檢測PD大鼠凋亡通路中TNF-α、P53、P21、Bcl-2/Bax、Casepase-3的表達(dá)變化；（3）改良大鼠大腦置管方法，在大鼠大腦的MFB部位埋植套管，通過套管給予6-OHDA造模PD模型大鼠。實驗動物分組：空白對照組(生理鹽水)，PD模型組(6-OHDA+5%DMSO)，藥物干預(yù)組(6-OHDA+Z-VAD-fmk)；行為學(xué)篩選出成功PD模型大鼠，藥物干預(yù)組將Caspase抑制劑Z-VAD-fmk通過套管注射入PD大鼠MFB中，每天一次。對照組及PD模型組每天給予等量5%DMSO（Z-VAD-fmk溶劑）；（4）形態(tài)學(xué)檢測：Nissl染色法觀察PD模型大鼠注射Z-VAD-fmk后神經(jīng)元丟失情況，電鏡觀察各種大鼠神經(jīng)細(xì)胞超微機(jī)構(gòu)的變化；（5）Real-time PCR檢測各組大鼠Bcl-2/Bax、RIP1、RIP3mRNA的表達(dá)變化。 結(jié)果：(1)注射6-OHDA2周后PD大鼠出現(xiàn)明顯的典型旋轉(zhuǎn)行為，Nissl染色及TH免疫組織化學(xué)染色標(biāo)記顯示PD模型大鼠毀損側(cè)黑質(zhì)致密部神經(jīng)元數(shù)量銳減（P0.01）；(2)與對照組相比，PD模型組黑質(zhì)及紋狀體中TNF-α、P53、P21、Bax、Casepase-3表達(dá)上調(diào)，，具有顯著差異（P0.05或P0.01），Bcl-2表達(dá)下調(diào)（P0.01）；(3)注射Caspase抑制劑Z-VAD-fmk后，PD大鼠模型動物行為學(xué)得到改善，黑質(zhì)神經(jīng)元丟失得到緩解，同時Bcl-2/Bax mRNA的表達(dá)上調(diào)；（4）電鏡結(jié)果顯示，Z-VAD-fmk在一定程度上能改善6-OHDA毀損所導(dǎo)致的線粒體減少、腫脹等超微結(jié)構(gòu)上的變化，但部分膠質(zhì)細(xì)胞出現(xiàn)水腫現(xiàn)象；（5）與模型組相比，藥物干預(yù)組黑質(zhì)RIP1，RIP3mRNA表達(dá)上升（P0.05），提示Z-VAD-fmk可能通過促進(jìn)活化的膠質(zhì)細(xì)胞壞死性凋亡（Necroptosis）而保護(hù)神經(jīng)元。 結(jié)論：（1）PD大鼠模型黑質(zhì)及紋狀體神經(jīng)細(xì)胞以程序性凋亡為主；（2）給予Z-VAD-fmk后，PD大鼠模型黑質(zhì)神經(jīng)細(xì)胞程序性凋亡在一定程度上被抑制，出現(xiàn)了膠質(zhì)細(xì)胞壞死性凋亡；（3）Z-VAD-fmk可能通過促進(jìn)活化的膠質(zhì)細(xì)胞壞死性凋亡而保護(hù)神經(jīng)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- 偽 -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- 偽 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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R742.5

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