【作者】 吴少波；

【导师】 武栋成；

【作者基本信息】 武汉大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 凋亡是神经系统发育中的正常生理现象。但在成年脑中,神经细胞的凋亡又成为某些急性和非急性神经退行性疾病如脑缺血、脑外伤、帕金森病和阿尔茨海默病等潜在的致病因素。已有研究表明,细胞周期素依赖性蛋白激酶-5（Cyclin-dependent kinase-5,Cdk5）的异常表达和活性失调与神经细胞的凋亡有关,但其作用机制仍未完全阐明。本文以神经细胞样分化的大鼠肾上腺嗜铬细胞瘤细胞系PC12细胞作为模型,以神经生长因子（nerve growth factor,NGF）撤退和星形孢菌素（Staurosporine,STS）诱导细胞凋亡,探讨Cdk5、p35及p53在PC12细胞凋亡中可能的作用机制。主要内容如下: 1.建立NGF撤退诱导的分化的PC12细胞凋亡模型,分析Cdk5/p35和p53的表达水平在此凋亡途径中的变化,探讨其可能的作用机制。 实验结果:MTT比色法可见,NGF撤退48h对PC12细胞MTT代谢有明显抑制作用,与对照组比较,差异有高度显著性（P<0.01）。Roscovitine和JNK抑制剂SP600125对NGF撤退造成的PC12细胞损伤均有保护作用（P<0.05）;突变型p53^R175H质粒的转染PC12对细胞有显著保护作用（P<0.01）。 琼脂糖凝胶电泳显示NGF撤退36h后有DNA凋亡梯带的产生。透射电镜观察可见,NGF撤退16h细胞出现染色质聚集,核膜缺损;36h后可见明显的凋亡形态学特征。流式细胞术检测显示NGF撤退24h后细胞凋亡率上升;SP600125和roscovitine使凋亡率显著下降;突变型p53^R175H的转染使凋亡率显著下降。Hoechst 33258染色可见,NGF撤退24h后细胞核呈典型的凋亡形态学特征:roscovitine预处理后核固缩细胞数目显著减少;SP600125的预处理使细胞受NGF撤退损伤程度减轻,核固缩细胞数目减少;转染突变型p53^R175H后,核固缩细胞数目显著减少,大多数细胞核形态正常。 Western blotting结果表明NGF撤退引起了Cdk5、p35、p53和p21蛋白表达的增加,且Cdk5表达增加早于p53蛋白。Roscovitine对Cdk5的表达水平无明显影响;SP600125使p53的表达水平有所降低,但对Cdk5的表达水平无明显影响;细胞转染突变型p53^R175H质粒后,p53的表达有显著增高,但Cdk5、p35的表达水平无明显变化。 2.建立STS诱导的分化的PC12细胞凋亡模型,分析Cdk5/p35和p53的更多还原

【Abstract】 There has been considerable interest in the molecular mechanisms of apoptosis in mammalian neurons because this form of neuronal cell death is important for the normal development of the nervous system and because inappropriate neuronal apoptosis may contribute to the pathology of human neurodegenerative diseases. It has been reported that, the deregulation of expression and activity of Cyclin-dependent kinase-5 （Cdk5） is involved in the neuronal apoptosis, but its mechanism has not been clarified completely yet. To investigate the role of Cdk5, p35 and p53 in the neuronal apoptosis, we established the apoptotic model of neuronal-differentiated rat adrenal pheochromocytoma cells （PC12） induced by nerve growth factor （NGF）- deprivation and staurosporine （STS）. The present study was performed in several parts as follows:1. Observed the changes of Cdk5 and p53 expression levels in apoptosis of PC12 cell induced by NGF-deprivation, and inquiring into the functional relation that may exist between Cdk5 and p53.The results displayed that NGF-deprivation resulted in a significant inhibition of MTT reduction in PC12 cells compared with control （P<0.01）; roscovitine inhibited the injury from NGF-deprivation （P<0.01）; JNK inhibitor SP600125 protected against apoptosis incompletely （P<0.05）; transfection p53^R175H plasmid into PC12 provided protective effects （P<0.01）. A "ladder" pattern representing fragmentation of DNA into oligonucleosome length fragments was observed after 48 h of NGF-deprivation. TEM analysis showed that cells deprivated NGF revealed prominent chromatin condensation, nuclear pyknosis and fragmentation, and followed other cytoplasmic alterations. The FCM shown that the rising apoptotic rates were detected on 36 h of lacking NGF; roscovitine made apoptotic rates significantly decrease; SP600125 decreased apoptotic rate partially; transfection of pBabe-p53^R175H decreased apoptotic rate significantly. Dyed by Hoechst 33258 showed typical nuclear morphological characteristics of apoptotic when NGF-deprivation 24 h, nuclei were severallyshrunken and compacted with condensed chromatin; in the presence of roscovitine, a significant reduction in number of the condensed nuclei was observed, and most of nuclei maintained their normal shape and size; pretreated with SP600125 reduced the number of the condensed nuclei during NGF-deprivation insults; transfection with p53R175H plasmid, the number of the condensed nuclei reduced markedly during NGF-deprivation insults. The results of western blotting displayed that NGF-deprivation increased Cdk5, p35, p53 and p21 expression, furthermore, Cdk5 protein expression were increased ahead of p53; roscovitine did not change the expression level of Cdk5; SP600125 made p53 expression lower; transfection pBabe-p53R175H into PC12 increased the p53 expression notably in both pathway, but no obvious influence to the expression level of Cdk5 and p35.2. Established the model of apoptotic neuronal PC12 cells induced by STS, and analyzed the role of Cdk5 /p35 and p53 protein in the apoptosis.The results manifestation that STS damaged PCI 2 cells in a dose-time dependent manner. Exposure to 300 nM STS resulted in a significant inhibition of MTT metabolism in PCI 2 cells compared with control （P<0.01）; Cdk5 inhibitor roscovitine protecting PC12 cells from STS （P<0.01）. Agarose gel electrophoresis of DNA fragmentation presented a "ladder" pattern after 6 h of STS treatment. Transmission electron microscopy （TEM） analysis showed that STS-treated cells revealed prominent chromatin condensation, nuclear pyknosis and fragmentation. Cytoplasmic alterations included disappearance of Golgi apparatus, dilation of the rough endoplasmic reticulum and mitochondria. The rising apoptotic rates were detected by flow cytometry （FCM） since 3 h of STS treatment; The FCM also showed that roscovitine made apoptotic rate indued by STS significantly decrease. The typical nuclear morphological characteristics of apoptotic were observed when exposure to STS 12h by staining Hoechst 33258, nuclei were severally shrunken and compacted with condensed chromatin; a significant reduction in number of the condensed nuclei was observed in the presence of roscovitine during STS insults, and most of nuclei maintained their normal shape and size. The results of western blotting displayed that STS increased Cdk5 and p35 expression, but had no effect on expression of p53;roscovitine did not change the expression level of Cdk5.To confirm the roles of Cdk5, p35 and p53 during the apoptosis of PC12 cells, we transfected Cdk5, p35, or Cdk5/p35 plasmids into cells. FCM showed increased apoptotic rates after transfection of these plasmids. And the number of the condensed nuclei markedly rised staining by Hoechst 33258; transfetion of Cdk5, p35, or Cdk5/p35 plasmids heighten the expression level of Cdk5 and p35 markedly, and made the p53 expression higher. Transfection of p53 plasmid into PC12 boosted the p53 expression notably, but no obvious changes of Cdk5 and p35 expression was observed.Conclusion: STS and NGF-deprivation could induce apoptosis in neuronal differentiated PC12 cells. The mechanism of STS-induced apoptosis might be related to the enhancement of Cdk5 and p35 expression and is p53-independent. NGF-deprivation induced apoptosis may involve in the enhancement of Cdk5, p35 and p53 expression, and p53 is probably the downstream effecter of Cdk5 activation. The effects of Cdk5 on apoptosis induced by STS or NGF-deprivation were not exactly identical, indicating that more than one effecters of Cdk5 may exist. And JNK may play a role in apoptosis of PC 12 cells induced by NGF-deprivarion.更多还原