体外缺血后处理对缺血再灌注损伤大鼠肾小管上皮细胞间质转变的影响

目的 探讨缺血后处理对肾小管上皮间质转变的影响.方法 应用NRK-52E建立有效的体外模型,实验分为对照组（COS）：应用对照缓冲液培养,3h后换完全培养基培养；缺血再灌注损伤组（IRI）：应用缺血缓冲液,3h后同样更换完全培养基培养；缺血后处理组（IPO）：应用缺血缓冲液培养3h,立即行体外缺血后处理,然后更换完全培养基.以上各组于处理3、6、12、24 h后收集细胞,Hoechst用于检测细胞凋亡,蛋白印迹法检测α-平滑肌肌动蛋白（α-SMA）、转化生长因子-β1（TGF-β1）、结缔组织生长因子（CTGF）的蛋白表达水平.结果 在经历缺血3h后再灌注24 h,IRI组和IPO组有明显细胞凋亡,但IPO组凋亡明显较IRI组减轻（P＜0.05）.IRI组α-SMA随时间延长表达逐渐增强,在24 h时表达最强（P＜0.05）；而IPO组随时间延长表达逐渐减弱,在24 h时达到最低值（P＜ 0.05）.IRI组TGF-β1在缺血再灌注3、6h时表达较强,之后减弱；且IPO组表达与IRI组相似,但3、6h时表达水平明显受到抑制.IRI组CTGF在缺血再灌注3、6h时表达较强,之后减弱；而IPO组表达各时间点较COS组没有明显变化.结论 体外缺血再灌注损伤可以诱导肾小管上皮细胞α-SMA的表达,诱导上皮细胞间质转化；而原因可能与激活TGF-β1,进而也抑制基质蛋白的降解,促进基质蛋白CTGF的表达等有关；同时IPO能有效抑制α-SMA、TGF-β1、CTGF的表达,说明其能抑制间质转化的发生.

The protective effects of ischemic postconditioning on renal tubular EMT after renal tubular epithelial ischemia-reperfusion injury in rats

Objective To study the effects of ischemia posteonditioning on renal tubular epithelial-mesenchymal tran- sition in vitro. Methods A valid in vitro ischemia postconditioning model was established by using NRK-52E cell line. Experiment was divided into three groups, the control group （COS）： application of control buffer cultured 3 h and then changed to complete medium; the ischemia -reperfusion injury （IRI） group： application of isehemic buffer, and 3 h later replaced to complete medium; ischemia postconditioning （IPO） group： ischemic buffer cultured 3 h, then imme- diately implemented postconditioning, and following change to the complete medium. Each group cells were collected at the reperfusion time of 3, 6, 12, 24 h, the cell apoptosis was detected by Hoechst, and the protein expression levels of α-SMA, TGF-131 and CTGF were detected by Western blot. Results After 3 h ischemia and 24 h reperfusion, the IRI and IPO groups had obvious apoptosis, but the apoptosis rate of IPO group was lower than the IRI group （P 〈 0.05）. The expression of cL-SMA in IRI group increased with time, and expressed to the strongest at 24 h （P 〈 0.05）. However, the expression gradually weakened with time in IPO group, and at 24 h expression to the lowest （P 〈 0.05）. The expression of TGF-31 in IRI group increased at point of 3, 6 h reperfusion, then decreased; interestingly, the IPO group expression similar to the IRI group, and the expression levels at 3, 6 h were significantly suppressed. Interesting- ly, the expression of CTGF in IRI group similar to TGF-β, the expression levels at 3, 6 h significantly increased, then decreased; compared with the COS group, the IPO group expression did not change significantly at each time point. Conclusion Ischemia-reperfusion injury in vitro can induce α-SMA expression in renal tubular epithelial cells, which can induce epithelial cell undergoing EMT; and the mechanism may be that it activates TGF-β1, and thus inhibits the degradation of matrix proteins, promotes expression of matrix protein CTGF and so on; while the most important is that IPO can effectively inhibit the expression of α-SMA, CTGF and TGF-β1, thus to inhibit the occurrence of EMT.