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人肾小管上皮细胞necroptosis模型的构建

发布时间:2019-01-04 19:11
【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(P0.05)。(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(P0.05)。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。
[Abstract]:Aim: to construct a model of apoptosis-like necrosis (necroptosis) of renal tubular epithelial cells (HK-2 cells) by using cultured human renal tubular epithelial cells (HK-2 cells) as target cells in vitro. Methods: tumor necrosis factor 伪 (tumor nercosis factor 伪 (TNF- 伪) was used to induce apoptosis and anti-mycin A (antimycin A) depletion ATP, was used to construct the model of renal tubular epithelial cell apoptosis. Caspase-8 inhibitor benzoxycarbonyl valeryl alanyl aspartyl fluoromethyl ketone (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone,zVAD-fmk) was used to block apoptosis and necrostatin-1 (Nec-1) a specific inhibitor of necroptosis was used to block necroptosis,. The morphological changes of cells were observed under different treatments. The cell survival rate and the expression of microtubule-associated protein 1 light chain 3- 鈪,

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