缺氧诱导因子1α在非离子造影剂诱导的肾小管上皮细胞凋亡中的作用及其机制  

Hypoxia-inducible factor-1α and renal tubular epithelial apoptosis induced by non-ionic iodinated contrast

作　　者：李慧凛[1] 张金元[2] 

机构地区：[1]南京军区总医院博士后科研工作站解放军第四五五医院博士后培养点,上海200052 [2]解放军第四五五医院南京军区肾脏病专科中心

出　　处：《肾脏病与透析肾移植杂志》2008年第4期339-346,共8页Chinese Journal of Nephrology,Dialysis & Transplantation

基　　金：中国博士后科学基金资助项目(20070421034)

摘　　要：目的:应用缺氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α)诱导剂氯化钴上调HIF-1α水平,观察其对非离子造影剂碘必乐诱导的HK-2细胞凋亡的作用和机制。方法:体外培养的HK-2细胞,分为阴性对照组、碘必乐阳性对照组(296mgI/ml,12h)、不同剂量及作用时间的氯化钴预处理与碘必乐共同作用组(氯化钴浓度为5、25、50、100μmol/L,分别预处理0h、1h、2h、4h、6h、12h)。通过流式细胞仪测定细胞凋亡率,Hoechst33258染色观察细胞凋亡的形态学变化,WesternBlot方法检测HIF-1α、Caspase-3和凋亡相关蛋白Bcl-2的表达水平。结果:(1)流式细胞仪检测结果表明,50μmol/L氯化钴预处理4h、6h和12h点与阳性对照组相比细胞凋亡率明显降低(10.95%,8.65%,8.61%vs22.06%,P<0.05),其中6h、12h点较4h点对细胞凋亡的抑制作用更强(P<0.05),而6h和12h点之间差异无统计学意义(P>0.05)。100μmol/L与50μmol/L氯化钴预处理相同时间(6h、12h)细胞凋亡率差异无统计学意义(P>0.05);(2)Hoechst33258染色结果发现,与阳性对照组相比,50μmol/L氯化钴预处理6h和12h凋亡细胞明显减少,其它时间点与阳性对照组差异无统计学意义。(3)Western Blot检测方法表明,50μmol/L氯化钴预处理1h以上即可引起HIF-1α表达增高,6h点表达最高(0.373±0.032),12h点较前下降(0.285±0.048)。与阳性对照组相比,50μmol/L氯化钴预处理4h、6h、12h,细胞内Caspase-3水平明显降低(P<0.05,0.346±0.037,0.294±0.062,0.179±0.051vs0.501±0.039)。Bcl-2表达水平明显增加(P<0.05,0.275±0.052,0.324±0.046,0.337±0.039vs0.099±0.025)。结论:HIF-1α对非离子造影剂碘必乐诱导的人近端肾小管上皮细胞凋亡有明显的抑制作用,其作用机制可能与下调Caspase-3和上调抗凋亡蛋白Bcl-2有关。Objective：Recent studies demonstrated that hypoxic injury of renal tubular cell played an important role in the pathogenesis of radiocontrast nephropathy. In this study, we investigated the role of hypoxia-inducible factor-1α （HIF-1α） upregulated by Cobalt Chloride （CoCl2 ） in iopamidol induced tubular cell apoptosis in vitro. Methodology： Cultured human proximal tubular epithelial cells （HK-2） were divided into three groups： negative control, positive control treated with iopamidol （296 mgI/ml, 12 h） and pretreated group which treated with CoCl2 （5,25,50 and 100 μmol/L） at different time points （0 h,1 h,2 h,4 h,6 h and 12 h） and then iopamidol （296 mgl/ml,12 h）. The proportion and morphologic change of apoptotic cell was examined by flow cytometry and Hoechst 33258 staining. The protein level of HIF-1α, caspase-3 and Bcl-2 in HK-2 cells was determined by Western blot analysis. Results：By flow cytometry, 50 μmol/L CoCl2 pre-treated for 4, 6 and 12 hours reduced the proportion of apoptotic cell significantly （P 〈 0. 05 ）. The proportion of apoptotic cells were lowered at 6 h and 12 h time points compared with 4 h time point （ P 〈 0. 05 ）. The proportion of apoptotic cells in CoCl2 pretreated for 6 and 12 hours was similar （ P 〉 0.05 ）. There was no significant difference in the proportion of apoptotic cell between 50 μmol/L and 100 μmol/L pretreated groups at the same time points （6 h and 12 h, P 〉 0. 05）. By Hoechst 33258 staining, 50 μmol/L CoCl2 pretreated for 6 and 12 hours attenuated the morphological changes of ceils compared with positive control. Western blot analysis revealed that the level of HIF-1α of HK-2 cells pretreated with 50 μmol/L CoCl2 over 1 hour was significantly increased （P 〈 0. 05 ）. The level of HIF-1α expression reached the peak value at the time point of 6 hours and then presented descending trend. Compared with positive control, the expression of caspase-3 of HK-2 ceils exposed to iopamidol and pretreated with

关 键 词：缺氧诱导因子1Α 造影剂肾病 氯化钴 非离子造影剂 细胞凋亡 

分 类 号：R692.6[医药卫生—泌尿科学]