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α(tumor nercosis factorα,TNF-α)诱导细胞凋亡,同时采用抗霉素A(antimycin A)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-缬氨酰-丙氨酰-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-associated protein 1 light chain 3-Ⅱ,LC3-Ⅱ)的表达。结果:(1)在TNF-α+zVAD-fmk+antimycin A处理1 h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-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+antimycin A1 h实验组,Nec-1预处理后细胞的存活率显著增加(P<0.05)。(3)TNF-α+zVAD-fmk+antimycin A干预1 h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-1能特异性阻断肾小管上皮细胞发生坏死。
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.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2013年第12期2301-2304,共4页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81170683)
