【作者】 朱冬菊；

【导师】 张玲；

【作者基本信息】 重庆医科大学 ， 内科学， 2020， 博士

【摘要】 第一部分缺血再灌注对肾小管上皮细胞肝再生增强因子的表达及线粒体自噬水平的影响目的:探讨体外缺血再灌注(ischemia reperfusion,I/R)处理对人肾小管上皮细胞(HK-2)肝再生增强因子(augmenter of liver regeneration,ALR)的表达及线粒体自噬水平的影响。方法:缺氧复氧与无糖无血清培养基相联合的方法对HK-2细胞进行处理,构建体外I/R模型。I/R处理时间设置为:缺氧6小时后再分别复氧3小时、6小时、12小时、24小时(I6R3、I6R6、I6R12、I6R24)。通过Western blot检测LC3-Ⅱ、TOMM20、TIMM23以及23kDa ALR的蛋白表达水平。通过电镜检测HK-2细胞经I6R12处理后线粒体形态学变化及线粒体自噬体的形成。结果:I/R处理后,23 kDa ALR及LC3-Ⅱ蛋白表达水平逐渐增高,在I6R12时间点增至最高,后下降;而TOMM20、TIMM23蛋白水平逐渐下降,在I6R12时间点降至最低,后有所增高。电镜结果显示I6R12处理后,HK-2细胞内线粒体肿胀明显,线粒嵴断裂,可见线粒体自噬体和自噬小体。结论:1.Western blot和电镜结果证实I/R处理成功诱导线粒体损伤和线粒体自噬现象的发生。在I/R处理过程中,HK-2细胞线粒体自噬水平先升高后降低,线粒体损伤在I6R12最明显,该时间点定为后续实验的观察点。2.23 kDa ALR参与了I/R诱导的线粒体自噬过程,其蛋白表达水平与LC3-Ⅱ变化一致,随时间延长而逐渐升高,在12h达到最高水平,随后逐渐下降。第二部份下调ALR表达对缺血再灌注诱导的HK-2细胞线粒体自噬水平的影响目的:建立稳定下调ALR表达的HK-2细胞株;探讨下调ALR表达对I/R诱导的线粒体自噬水平的影响。方法:慢病毒转染细胞后结合嘌呤筛选构建稳定下调23 kDa ALR表达的肾小管上皮细胞株。实验分组如下:ALR沉默缺血再灌注组(IR+shRNA/ALR组)、空载病毒缺血再灌注组(IR+shRNA/control组)、HK-2细胞缺血再灌注组(IR组)及HK-2细胞无处理组(Normal组)。q PCR检测ALR的m RNA表达水平。Western blot验证ALR的蛋白表达水平。LDH释放实验及细胞计数检测I/R处理对各组细胞的损伤情况。激光共聚焦显微镜观察LC3与线粒体的共定位情况。免疫印迹法检测ALR,线粒体自噬相关蛋白LC3-Ⅱ,p62,TOMM20,TIMM23及凋亡相关蛋白caspase-3的表达。流式细胞仪检测线粒体活性氧(reactive oxygen,ROS)水平。激光共聚焦显微镜及流式细胞仪检测线粒体膜电位变化。ATP检测试剂盒检测各实验组细胞内ATP含量的变化。流式细胞仪检测各实验组细胞凋亡率。结果:qPCR及免疫印迹检测显示,与shRNA/control组及正常组比较,shRNA/ALR组细胞内源性ALR m RNA水平及蛋白表达显著降低(P<0.05)。与shRNA/control组比较,I/R处理后shRNA/ALR组LDH释放量明显增高,而细胞数明显降低(P<0.05)。免疫荧光结果显示IR+shRNA/ALR组的LC3荧光信号与线粒体重叠明显减少。免疫印迹法检测结果显示,与IR+shRNA/control组及IR组比较,IR+sh RAN/ALR组ALR、LC3-Ⅱ蛋白水平降低,而P62、TOMM20和TIMM23以及cleaved CASP-3蛋白水平增高(P<0.05);与IR+shRNA/control组比较,IR+shRNA/ALR组线粒体膜电位及细胞内ATP水平下降,而线粒体ROS水平和细胞凋亡率显著增加(P<0.05)。结论:1.成功构建了稳定下调23 kDa ALR表达的人肾小管上皮细胞株。2.下调HK-2细胞23 kDa ALR表达可降低线粒体自噬水平,加重线粒体损伤,促进线粒体释放ROS增多,导致细胞凋亡水平增加;提示ALR对HK-2细胞的保护作用与增强线粒体自噬有关。第三部分下调ALR表达对线粒体自噬通路PINK1/Parkin的影响目的:探讨PINK1/Parkin在I/R诱导的线粒体自噬中的作用以及下调ALR表达对PINK1/Parkin通路的影响。方法:实验分组和造模方法同第二部分。I/R处理后,激光共聚焦显微镜检测PINK1和Parkin与线粒体的共定位情况;使用线粒体提取试剂盒提取线粒体蛋白验证各实验分组中PINK1和Parkin蛋白的表达水平。结果:I/R处理后,PINK1和Parkin蛋白在线粒体中的表达量增高(P<0.05),共聚焦显微镜结果显示PINK1和Parkin的荧光信号与线粒体重叠增加。下调HK-2细胞内23 kDa ALR表达后,与IR组和IR+shRNA/control组比较,PINK1和Parkin蛋白在线粒体中的表达量降低,同时激光共聚焦结果分析显示与线粒体重叠的共定位系数(Pearson’s correlation)降低(P<0.05)。结论:1.PINK1/Parkin通路在I/R诱导的线粒体自噬中被激活。2.下调23 kDa ALR表达后,PINK1/Parkin信号通路活性明显降低,23 kDa ALR调控线粒体自噬的机制与PINK1/Parkin信号通路有关。更多还原

【Abstract】 PART Ⅰ THE EFFECTS OF ISCHEMIA-REPERFUSION TREATMENT ON THE EXPRESSION OF AUGMENTER OF LIVER REGENERATION AND THE LEVEL OF MITOPHAGY IN HUMAN KIDNEY PROXIMAL TUBULAR CELLSObjective: To test the augmenter of liver regeneration(ALR)protein expression and the level of mitophagy in human kidney proximal tubular(HK-2)cells induced by ischemia-reperfusion(I/R)in vitro.Methods: HK-2 cells were treated by hypoxic/reoxygenation and phosphate/serum-free medium in vitro.The time point of I/R treatment was set as follows: reoxygenation for 3 hours,6 hours,12 hours and 24 hours after hypoxia for 6 hours(I6R3,I6R6,I6R12 and I6R24).The expression of LC3 II,TOMM20,TIMM23 and 23 kDa ALR in HK-2 cells were detected by immunoblotting.transmission electron microscopy(TEM)was used to observe the morphologic alterations of mitochondria and mitophagosomes in HK-2 cells.Results: In response to I/R treatment,the protein expression level of 23 kDa ALR and LC3-Ⅱ increased gradually,and increased to the highest at the time point of I6R12,and then decreased;while the protein level of TOMM20 and TIMM23 decreased gradually,and decreased to the lowest at the time point of I6R12,and then increased.After I/R treatment,mitochondrial swelling,loss of mitochondrial cristae,mitophagosomes and autophagosomes were apparent in HK-2 cells.Conclusion:1.The results of western blot and TEM showed that I/R treatment successfully induced mitochondrial damage and mitophagy.After I/R treatment,the level of mitophagy increased and was most obvious in I6R12,which was the observation point of subsequent experiments.2.23 kDa ALR was involved in the process of I/R-induced mitophagy,and its protein expression level was consistent with that of LC3-Ⅱ,which gradually increased with time,reached the highest level at 12 h,and then gradually decreased.PART Ⅱ THE EFFECT OF KNOCKDOWN OF ALR ON THE LEVEL OF MITOPHAGY CAUSED BY ISCHEMIA-REPERFUSION IN HK-2 CELLSObjective: To construct HK-2 cell line which stably down-regulating ALR.To investigate the role of down-regulation of ALR in the mitophagy induced by I/R.Methods: HK-2 cells were transduced with LV-ALR shRNA and LV-shRNA according to the manufacturer’s operations.After 72 h,the successfully transduced cells were selected with puromycin.The experimental groups were as follows: IR+shRNA/ALR group,IR+shRNA/control group,IR group and normal group.The ALR m RNA level was examined by quantitative real-time PCR(q PCR),and the ALR protein expression was tested by immunoblotting.The lactate dehydrogenase(LDH)cytotoxicity assay and the cell number count were used to test cell injury caused by I/R.The colocalization of LC3 and mitochondria in HK-2 cells was examined by laser scanning confocal microscopy.The protein level of ALR,LC3-Ⅱ,P62,TOMM20,TIMM23 and cleaved Caspase-3 were detected by Western blot.The levels of mitochondrial reactive oxygen species(ROS)were measured by flow cytometry.Mitochondrial membrane potential(MMP)was assessed using flow cytometry and confocal microscopy.The intracellular ATP levels of HK-2 cells was assessed using an ATP assay kit.The rate of cell apoptosis was tested by flow cytometry.Results: The results of immunoblotting analysis and q PCR showed that the ALR protein expression and ALR m RNA level in the shRNA/ALR group were significantly lower than that in the shRNA/control group and the normal group(P<0.05).the release of LDH in the shRNA/ALR group was clearly increased after I/R treatment.In addition,the number of cells in the shRNA/ALR group was significantly decreased after I/R compared with the shRNA/control(P<0.05).The colocalization of LC3 and mitochondria was decreased in the IR+shRNA /ALR group.The protein expression of ALR and LC3-Ⅱ in the IR+shRNA/ALR group were significantly lower than that of the IR+shRNA/control group,while the protein expression of P62,TOMM20,TIMM23 and cleaved Caspase-3 in the IR+shRNA/ALR group were significantly higher than that of the IR+shRNA/control group(p<0.05).The level of MMP and mitochondrial ROS in the IR+shRNA/ALR group decreased compared with IR+shRNA/control group,while the level of ROS and cell apoptosis were increased in the IR+shRNA/ALR group compared with IR+shRNA/control group(P<0.05).Conclusion: 1.HK-2 cell line which stably down-regulating 23 kDa ALR is successfully established.2.Knockdown of 23 kDa ALR in HK-2 cells can reduce the level of mitophagy,which leads to the aggravation of mitochondrial damage,the increase of ROS released by mitochondria,and finally the increase of cell apoptosis.Therefore,the protective effect of ALR on HK-2 cells is related to the enhancement of mitophagy.PART Ⅲ THE EFFECT OF KNOCKDOWN OF ALR ON THE LEVEL OF PINK1/PARKIN PATHWAYObjective: To investigate the role of PINK1/Parkin in I/R-induced mitophagy and the effect of down-regulating ALR expression on PINK1/Parkin pathway.Methods: The methods of group dividing and model making were the same as the second part.After I/R treatment,the colocalization of PINK1 and Parkin with mitochondria was detected by confocal microscopy.The mitochondria fraction was isolated by mitochondrial protein extraction kit,and the protein expression of PINK1 and Parkin in each experimental group were detected by Western blot.Results: After I/R treatment,immunoblot analysis showed that the expression of PINK1 and parkin extracted from mitochondrial protein increased(P<0.05),and the confocal microscopy results also showed that the fluorescence signals of PINK1 and parkin obviously overlapped with mitochondria.In response to I/R,HK-2 cells with ALR shRNA had a decrease in PINK1 and Parkin protein expression,as well as colocalization with mitochondria in immunofluorescence staining compared with cells with control shRNA(P<0.05).Conclusion: 1.PINK1/Parkin pathway is activated in I/R-induced mitophagy.2.Silencing of 23 kDa ALR suppressed the activation of PINK1/Parkin pathway via double immunofluorescent staining and Western blot.These results suggested that 23 kDa ALR may regulate mitophagy via the PINK1/Parkin pathway.更多还原