【作者】 张丽芳；

【导师】 苏津自；

【作者基本信息】 福建医科大学 ， 内科学（心血管病）， 2016， 硕士

【摘要】 【目的】探讨自噬在对比剂诱导的人肾小管上皮细胞系(HK-2)细胞凋亡的作用及阿托伐他汀预处理在对比剂诱导HK-2细胞凋亡中的影响。【方法】1.以HK-2细胞为研究对象,进行体外培养。分别加入不同浓度(0~160mg I/ml)碘海醇培养HK-2细胞4h,80mg I/ml碘海醇培养HK-2细胞不同时间(0~6h),采用Western-blot检测自噬相关蛋白LC3,Beclin1的表达,MTT法检测细胞活力,探讨碘海醇对自噬现象的影响。2.阿托伐他汀(ATO,10-7mol/L)预处理24h,然后加入碘海醇(80 mg I/ml)干预细胞4h,与碘海醇组(CM,80 mg I/ml)进行对比,采用Western-blot检测自噬相关蛋白LC3,Beclin1的表达,MTT法检测细胞活力,流式细胞仪检测细胞凋亡,观察阿托伐他汀预处理干预碘海醇诱导的HK-2细胞凋亡及细胞自噬的变化。3.将HK-2细胞分为六组:对照组(Control组)、碘海醇组(CM组)、阿托伐他汀预处理组(ATO+CM组)、空载体转染组(NC-si RNA+CM组)、Beclin1-si RNA+CM组和Beclin1-si RNA+ATO+CM组。阿托伐他汀预处理24h,碘海醇(80 mg I/ml)干预细胞4h,采用Western-blot检测自噬相关蛋白LC3,Beclin1的表达,MTT法检测细胞活力,流式细胞仪检测细胞凋亡,探讨自噬在阿托伐他汀预处理干预对比剂诱导人肾小管上皮细胞凋亡中的作用。【结果】1.与Control组相比,80mg I/ml碘海醇培养细胞4h自噬相关蛋白LC3,Beclin1蛋白表达水平均有明显升高(P<0.05),细胞凋亡率显著升高(P<0.05),细胞活力显著降低(P<0.05);ATO+CM组细胞自噬相关蛋白LC3,Beclin1蛋白表达水平较CM组上调(P<0.05),细胞凋亡率下降(P<0.05),细胞活力高于CM组(P<0.05)。2.与NC-si RNA+CM组相比,Beclin1-si RNA+CM组和Beclin1-si RNA+ATO+CM组自噬相关蛋白LC3(尤其LC3Ⅱ)、Beclin1表达均明显减少(P<0.05),ATO+Beclin1-si RNA+CM组与Beclin1-si RNA+CM组相比差异无统计学意义(P>0.05)。3.与NC-si RNA+CM组相比,Beclin1-si RNA+CM组和Beclin1-si RNA+ATO+CM组细胞凋亡率均明显升高(P<0.05),ATO+Beclin1-si RNA+CM组与Beclin1-si RNA+CM组相比差异无统计学意义(P>0.05)。4.与NC-si RNA+CM组相比,Beclin1-si RNA+CM组和Beclin1-si RNA+ATO+CM组细胞活力均受到明显抑制(P<0.05),ATO+Beclin1-si RNA+CM组与Beclin1-si RNA+CM组相比差异无统计学意义(P>0.05)。【结论】1.阿托伐他汀处理可以进一步上调自噬,挽救细胞活力,减轻碘海醇导致的HK-2细胞凋亡。2.干扰自噬Beclin1基因的表达可加重碘海醇诱导的HK-2细胞凋亡。3.干扰自噬Beclin1基因的表达可消除阿托伐他汀对碘海醇诱导的HK-2细胞凋亡的保护作用。更多还原

【Abstract】 [Objective] To investigate the Role of autophagy in contrast media induced renal tubular epithelial cell apoptosis and effects of Atorvastatin.[Methods]1.Human renal tubular epithelial cell(HK-2) was cultured as the research object in vitro.HK-2 cells were exposed to Iohesol at different concentration for 4h. HK-2 cells were exposed to Iohesol at 80 mg I/l for variable incubation times. The change of cell proliferation was detected by MTT assay.Using western blot to detect the expression of LC3 and Beclin1 to observe autophagy of HK-2 cell induced by contrast media.2.The cells were incubated with Atorvastatin(10-7mol/L) for 24 h and then exposed to Iohexol(80 mg I/L)for 4h. The change of cell proliferation was detected by MTT assay.The apoptosis level of HK-2 cells was examined by flow cytometry. The expression of LC3 and Beclin1 was examined by Western blotting.3.HK-2 cells were divided into six groups(control group, Iohexol group, Atorvastatin +Iohexol group, the NC- si RNA + Iohexol group, Beclin1 si RNA +Iohexol group,Beclin1-si RNA +Atorvastatin + Iohexol group). The cells were incubated with or without of Atorvastatin pretreatment and then exposed to Iohexol(80 mg I/L)for 4h that which is to investigate the role of autophagy in Atorvastatin pretreatment to protect the apoptosis of HK-2 cells induced by contrast media. The change of cell proliferation was detected by MTT assay. The apoptosis level of HK-2 cells was examined by flow cytometry. The expression of LC3 and Beclin1 was examined by Western blotting.[Results]1.CM group showed a lower survival rate(P < 0.05), a higher apoptosis rate(P < 0.01)and higher expression of LC3, Beclin1 proteins(P < 0.05) compared with the negative control group;ATO+CM group showed a higher survival rate(P < 0.05), a lower apoptosis rate(P < 0.01) and higher expression of LC3, Beclin1 proteins(P < 0.05)compared with the the CM groups(P < 0.05).2.Beclin1 si RNA + CM group and Beclin1 si RNA + ATO + CM group showed significantly lower expression of LC3 than NC- si RNA + CM group(P < 0.05).Whereas no difference was found in the expression of LC3 between ATO + Beclin1 si RNA + CM group compared with Beclin1- si RNA + CM group.3.Beclin1 si RNA + CM group and Beclin1 si RNA + ATO + CM group showed higher apoptosis rate(P < 0.05) than NC- si RNA + CM group. The different of apoptosis rate between ATO + Beclin1 si RNA + CM group compared with Beclin1- si RNA + CM group there were no statistical significance(P > 0.05).4.Beclin1 si RNA + CM group and Beclin1 si RNA + ATO + CM group showed lower survival rate compared with the NC- si RNA + CM group(P < 0.05).The different of cells proliferation between ATO + Beclin1 si RNA + CM and Beclin1 si RNA + CM group were no statistical significance(P > 0.05).[Conclusion]1. Atorvastatin can further enhance autophagy, increase the cell proliferation and reduce the apoptosis.2. Inhibition of Beclin1 gene expression increased the cell apoptosis from Iohexol induces.3. Inhibition of Beclin1 gene expression abrogated the protective effects of Atorvastatin on Iohexol-induced apoptosis on HK-2 cell.更多还原