论文部分内容阅读
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。
OBJECTIVE: To establish a model of necroptosis of renal tubular epithelial cells with human renal tubular epithelial cells (HK-2 cells) cultured in vitro as target cells. Methods: Apoptosis was induced by tumor necrosis factor α (TNF-α), at the same time, anti-mycin A (ATP) was used to induce the apoptosis of renal tubular epithelial cells and caspase-8 Inhibition of apoptosis by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a specific inhibitor of necroptosis necroptosis was blocked by necrostatin-1 (Nec-1) and the morphological changes of cells under different treatments were observed. The cell viability and the microtubule-associated protein 1 light chain 3-Ⅱ 3-II, LC3-II) expression. Results: (1) The cells and organelles were swollen when treated with TNF-α + zVAD-fmk + antimycin A for 1 h. Cell membrane fragmentation and round mitochondria under electron microscope were observed. The swelling and crest gradually became fuzzy, and a large number of autophagic bodies , While Nec-1 pretreatment cell necrosis than the control group significantly improved. (2) The survival rate of cells after Nec-1 pretreatment was significantly increased in TNF-α + zVAD-fmk + antimycin A1 h experimental group (P <0.05). (3) The expression of LC3-Ⅱ in Nec-1 pretreatment group was significantly decreased after TNF-α + zVAD-fmk + antimycin A treatment for 1 h (P <0.05). Conclusion: Apoptosis can induce tubular necroptosis in the apoptotic environment, and necrosis of renal tubular epithelial cells can be specifically blocked by the inhibitor Nec-1.