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目的:探讨核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)过表达对乙醇诱导下大鼠肝星状细胞系HSC-T6激活与增殖及I型胶原mRNA及蛋白表达水平的影响。方法:采用脂质体介导法对HSC-T6进行pEGFP-Nrf2重组质粒及pEGFP-N1空载质粒瞬时转染,将细胞分为正常对照组、乙醇刺激组、乙醇刺激+pEGFP-Nrf2质粒组和乙醇刺激+pEGFP-N1空载质粒组。采用RT-PCR及Western blotting方法对HSC-T6中Nrf2、Ⅰ型胶原及α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)mRNA及蛋白表达水平进行检测,采用MTT法对HSC-T6细胞增殖水平进行检测,采用流式细胞术对HSC-T6细胞周期分布进行检测。结果:(1)荧光显微镜下观察显示pEGFP-Nrf2质粒成功转染HSC-T6,转染后48h Nrf2 mRNA及蛋白表达水平较其余组显著升高(P<0.05)。(2)乙醇刺激组与乙醇刺激+pEGFP-N1空载质粒组之间细胞增殖水平、Ⅰ型胶原、α-SMA mRNA及蛋白表达水平差异无统计学意义(P>0.05),均明显高于正常对照组(P<0.05),细胞周期分布G1期比例下降,S期比例升高(P<0.05),而乙醇刺激+pEGFP-Nrf2质粒组细胞增殖水平及I型胶原、α-SMA mRNA及蛋白表达水平与乙醇刺激组及乙醇刺激+pEGFP-N1空载质粒组相比均显著下降(P<0.05),细胞周期分布G1期比例显著上升,S期比例显著下降(P<0.05),呈G1/S期阻滞。结论:Nrf2过表达可显著抑制乙醇对HSC-T6 Ⅰ型胶原及α-SMA mRNA及蛋白表达的促进作用,使HSC-T6细胞周期发生G1/S期阻滞,抑制乙醇诱导的HSC-T6增殖水平的升高,提示其对乙醇诱导的HSC-T6细胞活化具有负性调控作用。
AIM: To investigate the effects of over-expression of nuclear factor E2-related factor 2 (Nrf2) on the activation and proliferation of hepatic stellate cell line HSC-T6 and the expression of type I collagen mRNA and protein in rat hepatic stellate cells . Methods: HSC-T6 cells were transiently transfected with pEGFP-Nrf2 plasmid and pEGFP-N1 empty plasmid by liposome-mediated method. The cells were divided into normal control group, ethanol stimulation group, ethanol stimulation + pEGFP-Nrf2 plasmid group And ethanol stimulation + pEGFP-N1 empty plasmid group. The expression of Nrf2, type I collagen and α-smooth muscle actin (α-SMA) mRNA and protein in HSC-T6 were detected by RT-PCR and Western blotting. T6 cell proliferation was detected by flow cytometry to detect the cell cycle distribution of HSC-T6. Results: (1) Fluorescence microscopy showed that the expression of Nrf2 mRNA and protein was significantly up-regulated in HSC-T6 cells transfected with pEGFP-Nrf2 plasmid (P <0.05) 48 h after transfection. (2) There was no significant difference in the expression of collagen type Ⅰ, α-SMA mRNA and protein between ethanol-stimulated group and ethanol-stimulated + pEGFP-N1 empty plasmid group (P> 0.05) Compared with the normal control group (P <0.05), the cell cycle distribution decreased in G1 phase and increased in S phase (P <0.05), while the expression of type I collagen, α-SMA mRNA and pEGFP-Nrf2 Compared with the ethanol-stimulated group and the pEGFP-N1-empty vector group, the protein expression was significantly decreased (P <0.05). The cell cycle distribution of G1 phase was significantly increased, while the proportion of S phase was significantly decreased (P <0.05) G1 / S block. Conclusion: Overexpression of Nrf2 can significantly inhibit the promotion of HSC-T6 type Ⅰ collagen and α-SMA mRNA and protein expression by ethanol, arresting the G1 / S phase of HSC-T6 cells and inhibiting the growth of HSC-T6 induced by ethanol The level of HSC-T6 cells activated, suggesting that it has a negative regulation of ethanol-induced activation of HSC-T6 cells.