促红细胞生成素通过抑制GSK3β介导的Bax线粒体转位减少6-羟基多巴胺诱导的PC12细胞凋亡

发布时间：2018-03-29 14:14

本文选题：帕金森病　切入点：6-羟基多巴胺　出处：《苏州大学》2015年博士论文

【摘要】：第一部分6-羟基多巴胺(6-OHDA)诱导PC12细胞凋亡及其可能机制目的采用6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱导PC12细胞损伤建立帕金森病的体外模型,探讨GSK3β在6-OHDA诱导的凋亡途径中的具体作用及6-OHDA对PC12凋亡的可能作用机制。方法采用6-羟基多巴胺诱导PC12细胞损伤建立帕金森病的体外模型,通过MTT检测以及台盼蓝染料排除试验检测不同浓度的6-OHDA处理后的PC12细胞活性和生存率。利用免疫印迹法测定:1、总GSK3β和p-GSK3β水平;2、Bax和β-肌动蛋白水平,β-肌动蛋白用作细胞质内蛋白的内参对照;3、Bax和prohibition蛋白水平,prohibition蛋白用作线粒体内蛋白的内参对照。结果1、6-OHDA刺激24后呈剂量依赖性(10、20、50、100、200μm)地抑制PC12细胞的细胞活性及细胞存活率。6-OHDA在100μM时显著抑制PC12的活性和存活率。2、Western blot检测结果显示,6-OHDA处理呈时间依赖性地减少了GSK3β磷酸化。通过提取细胞线粒体和细胞质来检查Bax在细胞线粒体中存在的情况,结果显示6-OHDA处理后细胞质中Bax含量减少了,线粒体中Bax含量增加了,提示6-OHDA诱发了Bax的线粒体转位。结论6-OHDA显著降低了PC12细胞的活性,促进了PC12细胞凋亡,GSK3β在6-OHDA诱导的凋亡中起到一定作用,6-OHDA处理呈时间依赖性地减少GSK3β磷酸化,诱发了Bax的线粒体转位,Bax可从细胞溶质转位至线粒体中,引起线粒体功能障碍,进而导致细胞凋亡。第二部分促红细胞生成素(EPO)对6-羟基多巴胺诱导PC12细胞凋亡的保护作用及其机制目的通过研究促红细胞生成素对6-OHDA处理的PC12细胞的糖原合成酶激酶3β的磷酸化、Bax的线粒体转位、Bcl-2表达水平、半胱天冬酶3活性,探讨促红细胞生成素(EPO)抑制6-羟基多巴胺(6-OHDA)诱导的凋亡的机制。方法PC12细胞用TDZD-8(25μM)或用不同浓度的EPO(0-10U/m L)预处理,随后用100μM 6-OHDA孵育。采用MTT检测和台盼蓝排除试验测定不同处理后的细胞活性;免疫印迹法观察GSK3β磷酸化水平、总GSK3β水平、Bax在细胞内分布情况、抗增殖蛋白水平、Bcl-2以及β-肌动蛋白水平;Hoechst 33258染色法来估算凋亡PC12细胞的比例,使用半胱天冬酶3比色测定试剂盒系统估算半胱天冬酶3活性。结果1、EPO呈浓度依赖性地(浓度从1至10U/m L不等)降低6-OHDA诱导的生长抑制,GSK3β抑制剂4-苯基-2-甲基-1,2,4-噻二唑—3,5-二酮(TDZD8)显著逆转6-OHDA诱导的生长抑制。2、EPO和TDZD-8显著逆转6-OHDA对GSK3β磷酸化的抑制,也显著抑制6-OHDA诱导的Bax线粒体转位。3、6-OHDA不改变细胞质内的Bcl-2表达水平,而EPO和TDZD-8两者也不影响Bcl-2的细胞质内表达水平。但6-OHDA显著降低了Bcl-2的线粒体内表达水平,而加入EPO和TDZD-8后可逆转6-OHDA诱导的Bcl-2的线粒体内表达降低。4、EPO和TDZD-8抑制6-OHDA诱导的细胞凋亡,6-OHDA显著增加半胱天冬酶3活性,而EPO和TDZD则显著抑制半胱天冬酶3活性升高。结论EPO通过促进6-OHDA处理的PC12细胞的GSK3β的磷酸化、从而减少Bax的线粒体转位、以及阻止线粒体内Bcl-2水平的降低、降低Caspase 3活性,来抑制6-OHDA诱导的凋亡,进而发挥神经保护作用,提示EPO是一个很有潜力的神经保护药物。
[Abstract]:The first part 6- of 6-hydroxydopamine (6-OHDA) induced PC12 cell apoptosis and its possible mechanism by 6- hydroxydopamine (6-hydroxydopamine, 6-OHDA) injury model in vitro of Parkinson disease induced by PC12 cells, and investigate the possible mechanism of 6-OHDA GSK3 beta specific role in apoptosis pathway in 6-OHDA induced apoptosis in PC12. Methods 6- hydroxy dopamine to establish the model of Parkinson's disease in vitro injury induced by PC12 cells was detected by MTT and trypan blue dye exclusion test 6-OHDA after treated with different concentrations of PC12 cell activity and survival rate was determined by Western blot method: 1, total GSK3 and p-GSK3 beta beta level; 2, Bax and beta actin, beta actin as the cytoplasm protein control; 3, Bax and prohibition protein level, prohibition protein was used as a mitochondrial protein loading control. Results after 24 1,6-OHDA stimulation In a dose dependent manner (10,20,50100200 m) cell activity and cell inhibition of PC12 cell survival in 100 M significantly inhibited the activity of PC12 and.2 survival rate of.6-OHDA, Western blot assay showed that 6-OHDA treatment time dependently decreased GSK3 phosphorylation. Check in cell mitochondria Bax the extraction of mitochondria and cytoplasm, showed the decrease of Bax content after 6-OHDA treatment in the cytoplasm, the increase of Bax content in the mitochondria, suggesting that 6-OHDA induced mitochondrial translocation of Bax. Conclusion 6-OHDA significantly decreased the activity of PC12 cells and promote the apoptosis of PC12 cells, GSK3 beta play a certain role in apoptosis induced by 6-OHDA in the 6-OHDA treatment time dependently decreased GSK3 phosphorylation and induce mitochondrial translocation of Bax and Bax from the cytosol translocation to mitochondria, causing mitochondrial dysfunction, and To induce cell apoptosis. The second part of erythropoietin (EPO) to the protective effect and mechanism of 6- hydroxy dopamine induced apoptosis in PC12 cells by glycogen synthase kinase of erythropoietin on 6-OHDA treated PC12 cells 3 beta phosphorylation, mitochondrial translocation of Bax, expression of Bcl-2, caspase 3 activity, to investigate the effect of erythropoietin (EPO) inhibited 6- hydroxydopamine (6-OHDA) mechanism induced apoptosis. Methods PC12 cells with TDZD-8 (25 M) or with different concentrations of EPO (0-10U/m L) pretreatment, followed by 100 M. 6-OHDA was incubated with MTT method and trypan blue exclusion test cell activity after different treatment; observation of GSK3 phosphorylation level by Western blot, total GSK3 levels, the distribution of Bax in the cell, anti proliferation protein levels, Bcl-2 and beta actin levels; Hoechst 33258 was used to estimate the apoptosis of PC12 The proportion of cells, using the caspase 3 assay kit system estimation of caspase 3 activity. Results in 1, EPO in a concentration dependent manner (concentration ranging from 1 to 10U/m L) decreased 6-OHDA induced growth inhibition, GSK3 beta inhibitor 4- phenyl -2- methyl -1,2,4- two - 3,5- two with thiophene ketone (TDZD8).2 significantly reversed 6-OHDA induced growth inhibition, inhibition of EPO and TDZD-8 significantly reversed GSK3 phosphorylation of 6-OHDA, also significantly inhibited 6-OHDA induced Bax mitochondrial translocation of.3,6-OHDA does not change the expression level of Bcl-2 in the cytoplasm, while both EPO and TDZD-8 did not affect the expression level of Bcl-2 in the cytoplasm. But 6-OHDA decreased significantly the expression of Bcl-2 in mitochondria, reducing.4 reversed 6-OHDA induced Bcl-2 expression in mitochondria can join EPO and TDZD-8, EPO and TDZD-8 inhibited 6-OHDA induced apoptosis, 6-OHDA significantly increased caspase 3 activity, While EPO and TDZD inhibited caspase 3 activity increased. Conclusion EPO can promote 6-OHDA PC12 cells treated with GSK3 beta phosphorylation, thereby reducing mitochondrial translocation of Bax, and reduce the level of block Bcl-2 in mitochondria and reduce the activity of Caspase 3, to inhibit 6-OHDA induced apoptosis, and thus play a role in neural protection, suggesting that EPO is a potential neuroprotective drugs.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R742.5

【相似文献】