【摘要】：目的:探討3-甲基腺嘌呤(3-methyladenine,3-MA)抑制大鼠肝星狀細(xì)胞(HSC-T6)自噬后對HSC-T6細(xì)胞活化、增殖、凋亡、周期的影響及可能的作用機(jī)制。方法:體外培養(yǎng)大鼠肝星狀細(xì)胞HSC-T6,用不同濃度的3-MA處理,分為四個(gè)實(shí)驗(yàn)組,即空白對照組(0.0mmol/l),低濃度組(2.5mmol/l),中濃度組(5.0mmol/l),高濃度組(10.0mmol/l)。用MTT法觀察細(xì)胞增殖活力;用流式細(xì)胞技術(shù)檢測4組細(xì)胞的周期和凋亡情況;用蛋白印跡法(Western blot)分析不同濃度3-MA作用下各組HSC-T6細(xì)胞活化標(biāo)志α-平滑肌肌動(dòng)蛋白(α-SMA)、自噬標(biāo)志蛋白LC3Ⅱ、細(xì)胞周期蛋白Cyclin A、CyclinB1以及凋亡關(guān)鍵調(diào)節(jié)蛋白Bcl-2、BAX的表達(dá)變化。結(jié)果:不同濃度的3-MA作用于HSC-T6細(xì)胞24h后,3-MA實(shí)驗(yàn)組的HSC-T6細(xì)胞增殖明顯受到抑制,細(xì)胞凋亡率逐漸增高(均P0.05);S期及G2/M期細(xì)胞比例均明顯增加(均P0.05);Western blot顯示加入3-MA處理后HCS-T6細(xì)胞LC3Ⅱ、α-SMA、Cyclin A、CyclinB1、Bcl-2表達(dá)量均逐漸下降(均P0.05),BAX表達(dá)量逐漸上調(diào)(P0.05)。結(jié)論:(1)自噬抑制劑3-MA下調(diào)HSC-T6細(xì)胞的自噬水平,抑制LC3Ⅰ向LC3Ⅱ轉(zhuǎn)化,同時(shí)抑制HSCs的增殖和活化效應(yīng);(2)3-MA抑制HSC-T6細(xì)胞自噬后可分別通過下調(diào)Cyclin A、Cyclin B1蛋白水平誘導(dǎo)HSCs細(xì)胞周期阻滯在S期、G2/M期;(3)3-MA抑制HSC-T6細(xì)胞自噬后可通過上調(diào)促凋亡蛋白BAX表達(dá)和下調(diào)抗凋亡蛋白Bcl-2的表達(dá),升高BAX/Bcl-2的比率而誘導(dǎo)HSC-T6細(xì)胞凋亡。
[Abstract]:Aim: to investigate the effects of 3-methyladenine (3-methyladenine 3-MA) on the activation, proliferation, apoptosis and cell cycle of rat hepatic stellate cells (HSC-T6) after autophagy. Methods: cultured rat hepatic stellate cells (HSC-T6,) were treated with different concentrations of 3-MA. They were divided into four groups: blank control group (0.0mmol/l), low concentration group (2.5mmol/l), middle concentration group (5.0mmol/l) and high concentration group (10.0mmol/l). MTT assay was used to observe cell proliferation activity, flow cytometry was used to detect cell cycle and apoptosis in 4 groups. The expression of 偽 -smooth muscle actin (LC3 鈪，

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