中文摘要：

目的：以体外培养的人肾小管上皮细胞（HK-2细胞）为靶细胞，构建肾小管上皮细胞凋亡样坏死（necroptosis）的模型。方法：采用肿瘤坏死因子仅（tumornercosisfactora，TNF-α）诱导细胞凋亡，同时采用抗霉素A（antimycinA）耗竭ATP，构建肾小管上皮细胞凋亡的模型，并以caspase-8抑制剂苄氧羰酰一缬氨酰一丙氨酰一天冬氨酰-氟甲基酮（benzyloxycarbonyl．Val—Ala—Asp-fluoromethylketone，zVAD-fmk）阻断凋亡，用necroptosis的特异性抑制剂necrostatin-1（Nec-1）阻断necroptosis，观察细胞在不同的处理下形态学的变化，同时检测细胞存活率及标志物微管相关蛋白1轻链3-Ⅱ（microtubule-associatedprotein1lightchain3--Ⅱ，LC3-Ⅱ）的表达。结果：（1）在TNF--α＋zVAD-fmk＋antimycinA处理1h时细胞及细胞器膨胀，电镜下细胞膜碎裂，线粒体变圆、肿胀，嵴逐渐模糊，胞浆中出现大量自噬小体，而Nec一1预处理后细胞的坏死程度较对照组明显改善。（2）在TNF-α＋zVAD-fmk＋antimycinA1h实验组，Nec-1预处理后细胞的存活率显著增加（P〈0．05）。（3）TNF—d＋zVAD-fmk＋antimycinA干预1h实验组在Nec．1预处理后LC3-Ⅱ的表达量明显下降（P〈0．05）。结论：凋亡环境中阻断凋亡可以诱导肾小管上皮细胞necroptosis，抑制剂Nec一1能特异性阻断肾小管上皮细胞发生坏死。

英文摘要：

AIM ： To make a model of necroptosis in human renal tubular epithelial HK-2 cells. METHODS ： To induce necroptosis, HK-2 cells were treated with tumor necrosis factor ct （TNF-ct） followed by ATP depletion, and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone （zVAD-fmk） was added to block the activity of caspase-8. The morpho- logical changes of the cells were observed under light microscope and electronic microscope. The cell viability was detected by CCK-8 assay, and the marker of necroptosis was analyzed by Western blotting. RESULTS： In the cells treated with TNF-ct followed by zVAD-fmk and antimycin A for 1 h, the morphological changes including the cell and organdie infla- tion, and membrane fragmentation, with a large amount of autophagysome, were observed. However, these abnormalities were markedly attenuated after treatment with Nec-1. Meanwhile, the cell viability was also significantly improved after u- sing Nec-1. No similar variation was observed in other groups. In addition, the expression of LC3-II was significantly de- creased in Nec-1 ＋ TNF-ot ＋ zVAD-fmk ＋ antimycin A （1 h） group compared with control group. CONCLUSION： TNF- ot stimulation and energy depletion induce necroptosis in renal tubular epithelial cells. Nec-1 inhibits necroptosis in a caspase-independent pathway, and may have therapeutic potential to prevent and treat renal ischemia injury.