【摘要】：目的:以體外培養(yǎng)的人腎小管上皮細(xì)胞(HK-2細(xì)胞)為靶細(xì)胞,構(gòu)建腎小管上皮細(xì)胞凋亡樣壞死(necroptosis)的模型。方法:采用腫瘤壞死因子α(tumor nercosis factorα,TNF-α)誘導(dǎo)細(xì)胞凋亡,同時采用抗霉素A(antimycin A)耗竭ATP,構(gòu)建腎小管上皮細(xì)胞凋亡的模型,并以caspase-8抑制劑芐氧羰酰-纈氨酰-丙氨酰-天冬氨酰-氟甲基酮(benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone,zVAD-fmk)阻斷凋亡,用necroptosis的特異性抑制劑necrostatin-1(Nec-1)阻斷necroptosis,觀察細(xì)胞在不同的處理下形態(tài)學(xué)的變化,同時檢測細(xì)胞存活率及標(biāo)志物微管相關(guān)蛋白1輕鏈3-Ⅱ(microtubule-associated protein 1 light chain 3-Ⅱ,LC3-Ⅱ)的表達(dá)。結(jié)果:(1)在TNF-α+zVAD-fmk+antimycin A處理1 h時細(xì)胞及細(xì)胞器膨脹,電鏡下細(xì)胞膜碎裂,線粒體變圓、腫脹,嵴逐漸模糊,胞漿中出現(xiàn)大量自噬小體,而Nec-1預(yù)處理后細(xì)胞的壞死程度較對照組明顯改善。(2)在TNF-α+zVAD-fmk+antimycin A1 h實(shí)驗(yàn)組,Nec-1預(yù)處理后細(xì)胞的存活率顯著增加(P0.05)。(3)TNF-α+zVAD-fmk+antimycin A干預(yù)1 h實(shí)驗(yàn)組在Nec-1預(yù)處理后LC3-Ⅱ的表達(dá)量明顯下降(P0.05)。結(jié)論:凋亡環(huán)境中阻斷凋亡可以誘導(dǎo)腎小管上皮細(xì)胞necroptosis,抑制劑Nec-1能特異性阻斷腎小管上皮細(xì)胞發(fā)生壞死。
[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 偽 (tumor nercosis factor 偽 (TNF- 偽) 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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