摘要
目的:探讨自噬在低氧诱导人肾小管上皮细胞株(HK-2)凋亡中的作用。方法:培养HK-2细胞,根据培养环境氧浓度不同分为对照组(21%O_2)和低氧组(1%O2);根据低氧培养时间不同,低氧处理细胞进一步分为:低氧6 h亚组、低氧12 h亚组、低氧18 h亚组和低氧24 h亚组。Western blot检测HK-2细胞自噬相关蛋白Beclin1、LC3Ⅱ/LC3Ⅰ、P62和凋亡相关蛋白活化的天冬氨酸特异性半胱氨酸蛋白酶(cleaved-caspase3)表达水平,流式细胞仪检测HK-2细胞凋亡情况;采用低氧诱导稳定表达GFP-LC3的HK-2细胞18 h,在荧光显微镜下观察GFP-LC3自噬小体的变化;进一步采用低氧联合自噬诱导剂雷帕霉素(rapamycin,RP)或自噬抑制剂巴弗洛霉素A1(bafilomycin A1,BAf A1)处理HK-2细胞,细胞分为常氧对照组、常氧+雷帕霉素/或巴弗洛霉素组、低氧对照组、低氧+雷帕霉素/或巴弗洛霉素组。Western blot检测Beclin1、LC3Ⅱ/LC3Ⅰ、P62和cleaved-caspase3表达水平,流式细胞仪检测细胞凋亡情况。结果:与对照组(0.161±0.043)相比较,低氧12 h[(0.315±0.060),P=0.019]、18 h[(0.586±0.063),P=0.000]、24 h[(0.698±0.085),P=0.000]亚组HK-2细胞cleaved-caspase3蛋白表达明显增多;低氧12 h、18 h、24 h亚组HK-2细胞凋亡率分别为(23.633±5.346)%、(25.200±4.782)%、(38.567±8.046)%,较对照组(11.800±3.966)%明显增多(P=0.036,P=0.021,P=0.000)。同时,低氧18 h、24 h亚组HK-2细胞表达Beclin1蛋白[(0.557±0.071),(0.897±0.074)[较对照组(0.249±0.040)明显上调(P=0.001,P=0.000);低氧6 h、12 h、18 h、24 h亚组细胞表达LC3Ⅱ/LC3Ⅰ蛋白[(1.795±0.143),(2.873±0.078),(3.029±0.089),(3.735±0.059)]较对照组(1.322±0.191)明显上调(P=0.036,P=0.000,P=0.000,P=0.000);细胞表达自噬底物P62蛋白[(0.776±0.061),(0.602±0.119),(0.419±0.117),(0.384±0.068)]较对照组(0.957±0.035)明显下调(P=0.028,P=0.001,P=0.000,P=0.000);低氧组细胞GFP-LC3斑点计数相对值为(40.667±6.028),较对照组(18.667±5.508)明显增加(P=0.010)。雷帕霉素增加自噬后,低氧诱导的HK-2细胞凋亡(26.433±3.402)及cleaved-caspase3蛋白表达(0.265±0.066)较低氧对照组[(36.133±5.856),(0.358±0.060)]明显减少(P=0.012,P=0.000);巴弗洛霉素A1抑制自噬后,低氧诱导的HK-2细胞凋亡增多(49.233±3.412)及cleaved-caspase3蛋白表达(1.242±0.110)较低氧对照组[(25.933±3.650),(0.659±0.060)]明显增加(P=0.000,P=0.000)。结论:自噬在低氧诱导的HK-2细胞凋亡中起保护作用。
Objective：To investigate the effect of autophagy on the hypoxia-induced apoptosis in human renal proximal tubular cells （HK-2）. Methods：Human proximal tubular epithelial cells cultured in vitro were divided into the control group（21%O2） and hypoxia group（1%O2）, based on the oxygen concentration. HK-2 cells cultured in hypoxia condition were then divided into 6 h hypoxia sub- group, 12 h hypoxia subgroup, 18 h hypoxia subgroup and 24 h hypoxia subgroup. The expression of autophagy-related proteins, Beclinl, LC3 I1/LC3 I ,P62 and apoptosis-related protein,activated cleaved-caspase 3 were detected by Western blot analysis,and the apoptosis was analyzed by flow cytometry assay. HK-2 cells stably expressing GFP-LC3 were induced by hypoxia for 18 hours, and the alteration of LC3 autophagosomes was examined by immunofluorescence（IF） assay. Then HK-2 ceils were co-treated with hypoxia and autophagy inducer rapamycin or autophagy inhibitor Bafi-lomycin A1 ,and they were divided into normoxia control group,nor- moxia plus rapamycin/Bafilomycin A1 group,hypoxia control group, hypoxia plus rapamycin/Bafilomycin A1 group. The expression of Beclinl, LC3Ⅱ/LC3Ⅰ , P62 and cleaved-caspase3 were detected by Western blot analysis,while the apoptosis was analyzed by flow cytometry assay. Results：Compared with the control group（0.161± 0.043）,the cleaved-caspase3 expression in 12 h[（0.315 ± 0.060）, P=0.019], 18 h[（0.586± 0.063） ,P=0.O00] and 24 h hypoxia subgroup[（0.698± 0.085） ,P=0.000] increased significantly. The apopto- sis of HK-2 cells in 12 h[（23.633 ± 5.346）%], 18 h[（25.200± 4.782）%] and 24 h hypoxia subgroup[（38.567 ± 8.046）%] increased significantly as compared with the control group[（11.800 ±3.966）%]（P=0.036,P=0.021,P=0.000）. The Beclinl expression in 18 h [（0.557±0.071） ,P=-0.001] and 24 h hypoxia subgroup[（0.897 ± 0.074）,P=-0.000] were higher than that in the control group（0.249 ±0.04）. The LC3 11/LC3 I expression in 6 h[（ 1.795± 0.143） ,P=0.036], 12 h[（2.873±0.078） ,P=O.000], 18 h[（3.029 ± 0.089） ,P= 0.000] and 24 h hypoxia subgroup[（3.735 ± 0.059）,P=0.000] were higher than that in the control group（1.322 ± 0.191）. The P62 expression in 6 h[（0.776 ± 0.061 ）,P=-0.028], 12 h[（0.602 ±0.119）,P=-0.001], 18 h[（0.419±0.117）,P=O.O00] and 24 h hypoxia subgroup [（0.384 ± 0.068） ,P=-0.000] were lower than that in the control group（0.957 ± 0.035）. The counting value of GFP-LC3 puneta in HK- 2 cells in hypoxia group（40.667 ± 6.028） was obviously more than that in the control group（18.667± 5.508）（P=-0.010）. We also found that the treatment of the autophagy inducer rapamycin significantly reduced the hypoxia-induced apoptosis[（26.433±3.402）, P= 0.012] and cleaved-caspase3 expression[（0.265 ± 0.066）,P=0.000] of HK-2 cells compared with that in the hypoxia control group [（36.133± 5.856）, （0.358± 0.060）],while the treatment of the autophagy inhibitor Bafilomycin A1 significantly increased the hypox- ia-induced apoptosis[（49.233±3.412）,P=0.000] and cleaved-caspase3 expression[（1.242 ± 0.110） ,P=0.000] of HK-2 cells as com- pared to the hypoxia control group[（25.933±3.650）, （0.659± 0.060）]. Conclusion：Autophagy protects against the hypoxia- induced apoptosis of HK-2 cells.
作者
杨姗
李冠胜
陈雪梅
Yang Shan;Li Guansheng;Chen Xuemei(Department of Emergency, The First Affiliated Hospital of Chongqing Medical Universit)
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2018年第3期431-438,共8页
Journal of Chongqing Medical University
关键词
低氧
人肾小管上皮细胞
自噬
凋亡
hypoxia
human renal proximal tubular cell
autophagy
apoptosis
作者简介
杨姗，Email：907508930@qq．com，研究方向：急性肾损伤。;通信作者：陈雪梅，Email：1557451771@qq．com。
