基于不同研究方法探讨间歇低氧条件下自噬加重海马神经细胞损伤的机制

发布时间：2018-06-10 13:52

本文选题：OSAHS + SiRNA　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的阻塞性睡眠呼吸暂停低通气综合征(OSAHS)可导致或加重认知功能障碍,其机制与间歇低氧(IH)所致海马神经细胞凋亡有关。我们前期研究初步发现间歇低氧可能是通过HIF-1α-BNIP3-Beclin1介导自噬发生,且自噬激活加重了海马神经细胞凋亡。但前期研究主要依赖于免疫印迹、体外药物干预的方法去探讨,由于任何单一一种检测自噬方法均不能准确的反映细胞自噬水平,应用多种实验方法检测自噬发生及其分子机制是对整个研究的进一步完善和升华。因此,本研究通过建立OSAHS体外细胞模型,应用免疫荧光和透射电子显微镜观察的实验方法从形态学角度检测间歇低氧条件下自噬的表达,通过基因沉默和免疫共沉淀的实验方法进一步明确间歇低氧条件下HIF-1α介导自噬发生的具体机制,并应用基因沉默和药物干预的实验方法初步探讨自噬激活加重海马神经细胞损伤的分子机制。方法(1)研究对象:购买出生24小时之内的SD乳鼠,培养原代海马神经元7-9天。(2)建立OSAHS细胞模型并根据实验不同条件进行分组:正常对照组(NC)、不同时间段的间歇低氧(IH)组。检测指标:免疫荧光技术检测自噬标记蛋白LC3-II的表达;透射电子显微镜下观察双层膜结构的自噬体的形成;(3)Si RNA转染与分组:给予Si RNA干预HIF-1α(Beclin1)表达,设阴性对照组、IH组、HIF-1αSi RNA(Beclin1 Si RNA)干预组、IH+HIF-1αSi RNA(Beclin1 Si RNA)干预组。选取HIF-1α及自噬表达变化最明显的间歇低氧时间点12h进行处理,HIF-1α(Beclin1)si RNA预处理方法:细胞培养7天给予HIF-1α(Beclin1)si RNA转染处理48h。检测指标:疫印迹技术检测各组海马神经细胞LC3-II、cleaved-caspase-3、HIF-1α-BNIP3-Beclin1信号通路相关蛋白的表达。(4)免疫共沉淀技术检测Beclin1-Bcl2的结合状态。(5)给予自噬抑制剂和激活剂干预已间歇低氧处理的海马神经细胞。分组为:正常对照组、间歇低氧组、药物预处理组、单独给药组。检测指标:免疫印迹技术检测各组海马神经细胞cleaved-caspase-3和XIAP蛋白的表达变化。结果(1)免疫荧光检测自噬的表达:随着间歇低氧时间延长,IH组LC3-II荧光点表达增多,且在IH12h组LC3-II表达最多,与NC组相比,差异具有统计学意义(P0.05)。(2)透射电子显微镜观察自噬体的形成:与NC组相比,IH组双层膜结构的自噬溶酶体明显增多。(3)Si RNA干预HIF-1α表达对海马神经元的自噬水平影响:免疫印迹结果显示,与NC组相比,IH组HIF-1α、LC3-II蛋白表达增多(P0.05);Si RNA+IH组HIF-1α和LC3-II表达较IH组明显减少(P0.05)。(4)Si RNA干预海马神经元HIF-1α表达后,检测HIF-1α、BNIP3和Beclin1蛋白的表达。免疫印迹结果显示,IH组HIF-1α、BNIP3、Beclin1蛋白表达较NC组明显增高(P0.05);Si RNA+IH组HIF-1α、BNIP3、Beclin1蛋白表达较IH组明显减少(P0.05)。(5)免疫共沉淀检测Bcl2和Beclin1的结合状态:NC组Beclin1和Bcl2处于结合状态,IH和EBSS组Beclin1和Bcl2处于分离状态。(6)间歇低氧对XIAP表达的影响:随着间歇低氧时间延长,IH组LC3-II和cleaved caspase-3蛋白表达呈增多趋势,而XIAP蛋白表达减少;IH12h组LC3-II和cleaved caspase-3蛋白表达最多而XIAP蛋白表达最少,与NC组相比,差异具有统计学意义(P0.05);(7)药物干预自噬后对XIAP蛋白和凋亡表达的影响:与IH组相比,RAP+IH组XIAP的表达明显减少,而LC3-II和cleaved caspase-3表达显著高于IH组(P0.05);CQ+IH组与IH组相比,在LC3-II和cleaved caspase-3表达减少的同时,XIAP表达增多,且差异均具有统计学意义(P0.05)。结论本研究证实:(1)从形态学角度进一步证明间歇低氧处理后海马神经细胞自噬表达增多;(2)间歇低氧状态下通过si RNA抑制HIF-1α的表达后,自噬标记蛋白LC3-II表达也降低;(3)间歇低氧状态下通过si RNA抑制HIF-1α的表达后HIF-1α-BNIP3-Beclin1信号通路蛋白表达均降低。(4)常氧状态下Beclin1以Beclin1-Bcl2复合体的形式存在于细胞中,而间歇低氧可促使Beclin1-Bcl2的分离释放出游离Beclin1以促进自噬的发生。(5)间歇低氧状态下通过Si RNA抑制Beclin1的表达后,促凋亡蛋白cleaved caspase-3表达也降低。(6)随着间歇低氧时间延长,自噬和凋亡增多,而凋亡抑制剂XIAP的表达减少。(7)通过药物抑制(促进)自噬表达后,在促凋亡蛋白cleaved caspase-3表达减少(增多)的同时,凋亡抑制剂XIAP蛋白的表达增多(减少)。综上所述,我们通过免疫荧光、透射电子显微镜、基因沉默、免疫共沉淀等实验方法对前期研究进行了完善和补充,结果证实:间歇低氧条件下HIF-1α通过激活其下游靶蛋白BNIP3促进了Beclin1-Bcl2的分离,所释放出的游离的Beclin1激活自噬并可能通过过度清除XIAP加重了海马神经细胞凋亡。
[Abstract]:Objective obstructive sleep apnea hypopnea syndrome (OSAHS) can lead to or aggravate cognitive dysfunction. Its mechanism is related to the apoptosis of hippocampal neurons induced by intermittent hypoxia (IH). Our preliminary study preliminarily found that intermittent hypoxia may be mediated by HIF-1 alpha -BNIP3-Beclin1, and autophagy activates the hippocampal neurons. But the previous study mainly depends on the immunoblotting and the methods of drug intervention in vitro. Because any single method of autophagy can not accurately reflect the level of autophagy, the application of a variety of experimental methods to detect the occurrence of autophagy and its molecular mechanism is the further improvement and sublimation of the whole research. The expression of autophagy was detected by the experimental method of immunofluorescence and transmission electron microscopy. The specific mechanism of HIF-1 alpha mediated autophagy under intermittent hypoxia was further clarified by the experimental method of immunofluorescence and transmission electron microscopy. The specific mechanism of autophagy mediated by OSAHS was further clarified. The molecular mechanism of autophagy activation aggravated the damage of hippocampal neurons was preliminarily investigated by the experimental methods of silence and drug intervention. Methods (1) the study object: to buy SD milk rats within 24 hours of birth and to cultivate the primary hippocampal neurons for 7-9 days. (2) the OSAHS cell model was established and grouped according to the different conditions: normal control group (NC), not simultaneously. Interintermittent hypoxia (IH) group. Detection index: the expression of autophagy protein LC3-II was detected by immunofluorescence; the formation of autophagic in the double layer membrane structure was observed under transmission electron microscope; (3) Si RNA transfection and grouping: Si RNA intervention was given to HIF-1 alpha (Beclin1) expression, negative control group, IH group, HIF-1 alpha Si RNA group intervention group, +HIF-1 alpha Si RNA (Beclin1 Si RNA) intervention group. Selected HIF-1 alpha and the most obvious intermittent hypoxia time point 12h to be treated, HIF-1 alpha (Beclin1) Si RNA preconditioning method: cell culture for 7 days. Se-3, HIF-1 alpha -BNIP3-Beclin1 signaling pathway related protein expression. (4) immuno coprecipitation technique to detect the binding state of Beclin1-Bcl2. (5) the intervention of autophagic inhibitors and activators to interfere with the intermittent hypoxia treatment of hippocampal neurons. Group: normal control, intermittent hypoxia group, drug preconditioning group, individual drug delivery group. The expression of cleaved-caspase-3 and XIAP protein in the hippocampal neurons of each group was detected by Western blot. Results (1) the expression of autophagy detected by immunofluorescence: the expression of LC3-II fluorescence points in group IH increased with the prolongation of intermittent hypoxia time, and the most expression in IH12h group LC3-II, compared with the NC group, the difference was statistically significant (P0.05). (2) transmission electron microscopy The autophagosome formation was observed by microscope: compared with the NC group, the autophagic lysosomes in the IH group were significantly increased. (3) the effect of HIF-1 alpha expression on the autophagy level of the hippocampal neurons was influenced by the intervention of Si RNA. The result of immunoblotting showed that the expression of HIF-1 A and LC3-II protein in the IH group increased (P0.05) compared with the NC group, and the Si RNA+IH group was significantly lower than that of the NC group. (4) (4) (4) Si RNA interfered with the expression of HIF-1 alpha in hippocampal neurons, and the expression of HIF-1, BNIP3 and Beclin1 protein was detected. The results of immunoblotting showed that the expression of HIF-1 alpha, BNIP3, Beclin1 protein in IH group was significantly higher than that of NC group. The binding state of eclin1: the NC group Beclin1 and Bcl2 are in a binding state, Beclin1 and Bcl2 in IH and EBSS groups are in separate state. (6) the effect of intermittent hypoxia on XIAP expression: with the prolongation of intermittent hypoxia, the expression of LC3-II and cleaved proteins in IH group increased and the expression of proteins decreased. The expression of XIAP protein was the least, and the difference was statistically significant compared with the NC group (P0.05). (7) the effect of the drug intervention on the expression of XIAP protein and apoptosis after autophagy: compared with the IH group, the expression of XIAP in the RAP+IH group was significantly reduced, and the expression of LC3-II and cleaved caspase-3 was significantly higher than that in the IH group (P0.05). The expression of ved caspase-3 was decreased and the expression of XIAP increased, and the difference was statistically significant (P0.05). Conclusion: (1) further evidence of the increase of autophagy expression in hippocampal neurons after intermittent hypoxia treatment was confirmed from the morphological point of view; (2) the expression of autophagy protein LC3-II table in the intermittent hypoxic state after the expression of HIF-1 alpha by Si RNA (3) the expression of HIF-1 alpha -BNIP3-Beclin1 signaling protein was reduced after Si RNA inhibition of HIF-1 alpha in intermittent hypoxia. (4) Beclin1 was in the form of Beclin1-Bcl2 complex in the normal oxygen state, and intermittent hypoxia could induce the separation and release of Beclin1-Bcl2 to promote the occurrence of autophagy. 5) the expression of apoptotic protein cleaved caspase-3 decreased after Si RNA inhibited Beclin1 expression in intermittent hypoxia. (6) the expression of autophagy and apoptosis increased with the prolongation of intermittent hypoxia, and the expression of apoptosis inhibitor XIAP decreased. (7) the expression of apoptotic protein cleaved caspase-3 decreased by drug inhibition (promoting) autophagic expression. At the same time, the expression of apoptosis inhibitor XIAP protein increased (decrease). In summary, we perfected and supplemented the previous studies by immunofluorescence, transmission electron microscopy, gene silencing, immunoprecipitation and other experimental methods. The results confirmed that HIF-1 alpha under intermittent hypoxia activates its downstream target protein BNIP3 to promote Beclin1- The free Beclin1 released by Bcl2 activates autophagy and may aggravate hippocampal neuronal apoptosis through overcleaning XIAP.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R766

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