ATRA对缺糖缺氧损伤PC12细胞的抗凋亡作用及机制探索

发布时间：2018-06-09 15:09

本文选题：缺糖缺氧损伤 + 全反式视黄酸　；参考：《重庆医科大学》2012年硕士论文

【摘要】：背景维生素A缺乏（VitaminAdeficiency, VAD）、铁缺乏、碘缺乏已被WHO和联合国儿童基金会确定为世界三大营养素缺乏性疾病。VAD在世界范围内普遍存在，其中孕妇和儿童是其高危人群。新生儿缺氧缺血性脑损伤(hypoxic-ischemic brain damage，HIBD)是新生儿期常见的中枢神经系统高发病、危重病，是导致新生儿死亡和残疾的主要原因之一。有研究表明，HIBD的主要发病机制包括脑血流动力学变化、脑细胞能量代谢衰竭、再灌注损伤与氧自由基的产生、钙内流、兴奋性氨基酸导致神经毒性作用、迟发性神经元死亡和凋亡，其中细胞凋亡是HIBD神经细胞死亡的主要形式。本课题组的前期研究发现，HIBD新生大鼠体内VA水平与其神经功能的修复存在密切的相关性，但其具体分子机制目前还不清楚。目的体外探讨全反式视黄酸（ATRA）对缺氧缺糖损伤（OGD）后的PC12细胞的修复作用。方法建立PC12细胞OGD损伤模型，给予不同ATRA浓度（0.5、2、4和20μmol/L）诱导，利用Annexin V-PI流式细胞术检测PC12细胞的凋亡发生率，荧光探针JC-1测定细胞线粒体膜电位的变化，，同时运用Real-time PCR技术和Western blot技术分析ATRA对OGD损伤后的PC12细胞线粒体凋亡通路关键性凋亡因子表达水平的影响。结果光镜下观察显示PC12细胞OGD损伤后，细胞聚团生长，胞体皱缩，折光性下降；Annexin V-PI、JC-1检测显示细胞凋亡率明显增加，线粒体膜电位下降明显；加入2、4μmol/LATRA后均可降低细胞凋亡率，并保持线粒体膜电位的相对稳定，尤以4μmol/LATRA作用效果明显；与对照组相比较，0.5μmol/L ATRA组没有显著差异；但20μmol/L ATRA组的细胞凋亡率及线粒体膜电位下降率增高。Real-timePCR和Western blot检测显示，4μmol/L ATRA组较OGD损伤组中的促凋亡因子Bax的表达水平显著下调，抗凋亡因子Bcl-2明显升高，蛋白表达水平与mRNA水平变化一致。结论4μmol/L ATRA对OGD损伤后的PC12细胞具有抑制凋亡的作用，其机制可能是ATRA通过上调Bcl-2和下调Bax的表达，影响线粒体凋亡通路，从而实现对缺糖缺氧损伤细胞的保护作用。背景HIBD后神经元的死亡形式主要为细胞凋亡，位于线粒体的Bcl-2家族及其编码的蛋白质表达失衡是线粒体凋亡通路中的关键事件，最终导致细胞结构和功能的广泛损害。本课题组前期的研究表明，体内正常VA水平更有效地促进HIBD大鼠的神经细胞的功能恢复，其机制可能是由于VAD抑制了RAR的表达，降低了神经细胞Ca~(2+)的兴奋性，从而影响学习记忆能力。我们前一部分研究结果显示，4μmol/LATRA能够通过调节线粒体凋亡通路，抑制神经细胞的凋亡，其机制是否是通过RAR的调节而发挥作用，将作进一步研究。目的探讨RA信号通路中RAR在缺糖缺氧损伤的PC12细胞抗凋亡的调节作用。方法采用本课题组自行构建的视黄酸核受体siRAR重组腺病毒（Ad-siRAR）感染PC12细胞，应用RT-PCR方法检测Ad-siRAR对RAR受体的抑制效率。利用Ad-siRAR技术，进一步观察4μmol/LATRA对损伤的PC12细胞抗凋亡的影响，同时以空腺病毒载体为对照。Annexin V-PI和荧光探针JC-1染色，流式细胞技术分别检测siRAR感染PC12细胞后细胞凋亡率及线粒体膜电位下降率的变化，进一步运用Real-time PCR和Western blot技术分析RAR受体与线粒体凋亡通路中关键因子基因和蛋白表达水平的变化。结果PC12细胞缺糖缺氧损伤后，RAR mRNA表达水平降低。Ad-siRAR感染36h后，可有效抑制RAR基因表达。经Annexin V-PI和荧光探针JC-1染色，流式细胞技术检测显示，4μmol/LATRA诱导的OGD损伤PC12细胞在Ad-siRAR感染条件下，其细胞凋亡率及线粒体膜电位下降率均明显增高。同时，Real-time PCR和Western blot结果表明，感染Ad-siRAR重组腺病毒的PC12细胞OGD损伤后，Bax表达水平显著增高，Bcl-2的表达降低。结论重组腺病毒Ad-siRAR显著抑制了ATRA对OGD损伤的PC12细胞的抗凋亡保护作用，提示ATRA通过调节RA信号通路中的RAR，影响线粒体凋亡信号通路中关键因子Bax和Bcl-2的表达水平，从而发挥对OGD损伤的PC12细胞的保护作用。
[Abstract]:Background Vitamin A deficiency ( VAD ) , iron deficiency , iodine deficiency have been identified by WHO and UNICEF as the world ' s three major nutritional deficiencies . VAD is prevalent worldwide , among which pregnant women and children are high - risk populations . The study shows that the main pathogenesis of HIBD includes brain hemodynamic changes , brain cell energy metabolism failure , reperfusion injury and oxygen free radical generation , calcium influx , excitatory amino acids leading to neurotoxicity , delayed neuronal death and apoptosis .

Objective To investigate the effect of all - trans retinoic acid ( ATRA ) on PC12 cells after hypoxia and glucose deprivation ( OGD ) .

Methods PC12 cell OGD injury model was established and induced by different concentrations of ATRA ( 0.5 , 2 , 4 and 20 渭mol / L ) . The apoptosis rate of PC12 cells was determined by flow cytometry . The effects of ATRA on the expression level of apoptosis in PC12 cells after OGD injury were analyzed by Real - time PCR and Western blot .

Results After the PC12 cell OGD injury was observed under the light microscope , the cell clusters were grown , the cell bodies shrink and the refractive index decreased ;
The apoptosis rate of the cells was significantly increased and the mitochondrial membrane potential decreased .
After addition of 2,4 - 渭mol / LATRA , the apoptosis rate was decreased , and the relative stability of mitochondrial membrane potential was maintained , especially in 4 渭mol / LATRA .
Compared with the control group , there was no significant difference in 0.5 渭mol / L ATRA group .
However , the apoptosis rate and mitochondrial membrane potential decreased significantly in the 20 渭mol / L ATRA group . Real - time PCR and Western blot showed that the expression level of pro - apoptotic factor Bax was down - regulated in 4 渭mol / L ATRA group compared with that in OGD group , and the expression level of anti - apoptotic factor Bcl - 2 increased significantly , and the level of protein expression was consistent with that of mRNA level .

Conclusion 4 渭mol / L ATRA can inhibit the apoptosis of PC12 cells after OGD injury . The mechanism may be that ATRA can regulate the expression of Bcl - 2 and down - regulate Bax and influence the apoptosis pathway of mitochondria .

Background : The death form of neurons in HIBD is mainly apoptosis . The expression imbalance of Bcl - 2 family and its encoded protein in mitochondria is a key event in the apoptosis pathway of mitochondria , which results in the extensive damage of cell structure and function .

Objective To investigate the effect of RARs on anti - apoptosis of PC12 cells induced by glucose deprivation and hypoxia .

Methods The effect of 4 渭mol / LATRA on the anti - apoptosis of PC12 cells was investigated by RT - PCR . The effects of 4 渭mol / LATRA on the anti - apoptosis of PC12 cells were investigated by RT - PCR .

Results After 36 hours of Ad - sirar infection , the apoptosis rate and mitochondrial membrane potential of PC12 cells infected with Ad - sirar were significantly increased , and the expression of Bax was significantly increased and the expression of Bcl - 2 decreased .

Conclusion The effect of ATRA on the anti - apoptotic effect of ATRA on PC12 cells induced by OGD was significantly inhibited by recombinant adenovirus Ad - sirar , suggesting that ATRA could play a role in protecting PC12 cells injured by OGD .
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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