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    线粒体内膜转位酶13调控肝星状细胞活化和增殖的实验研究

    Experimental study of translocase of inner mitochondrial membrane 13 regulating the activa⁃tion and proliferation of hepatic stellate cells

      摘要
    • 摘要: 目的：研究线粒体内膜转位酶13（TIMM13）的表达对小鼠肝星状细胞活化和增殖的影响，探讨TIMM13在肝纤维化的的进展中的作用。方法：用5 ng/mL、10 ng/mL、20 ng/mL 转化生长因子-β1（TGF-β1）作用小鼠肝星状细胞系JS-1 细胞24 h，诱导JS-1 细胞活化，实验分为对照组（0 ng/mL 组）、5 ng/mL 组、10 ng/mL 组、20 ng/mL 组；采用实时荧光定量PCR（RT-qPCR）和蛋白质印迹（western blotting）法检测各组TIMM13、肝纤维化指标α-平滑肌肌动蛋白（α-SMA）和Ⅰ型胶原蛋白（COL1A1）的mRNA 与蛋白表达水平；在JS-1 细胞中分别敲低和过表达TIMM13，将实验分为空载组（对照组）和沉默组/过表达组，用RTqPCR和western blotting法检测TIMM13、α-SMA和COL1A1的mRNA与蛋白表达水平，用细胞计数试剂盒（CCK-8）检测沉默/过表达TIMM13 的第0、第24、第48 和第72 小时细胞的增殖情况。结果：与对照组比较，5 ng/mL 组、10 ng/mL 组、20 ng/mL 组中TIMM13 的mRNA 表达升高（均P< 0.05），5 ng/mL 组、10 ng/mL 组中TIMM13 蛋白表达水平增高（均P< 0.05）；沉默组中TIMM13、α-SMA 和COL1A1 的mRNA 表达水平相对于对照组降低（均P< 0.01），TIMM13 和α-SMA 的蛋白表达水平降低（均P< 0.01），在第48小时和第72小时JS-1细胞增殖能力被明显抑制（均P< 0.05），而过表达组中TIMM13、α-SMA 和COL1A1的mRNA表达水平升高（均P< 0.05），TIMM13和α-SMA的蛋白表达水平升高（均P< 0.05），在第48 小时和第72小时促进了JS-1细胞的增殖能力（均P< 0.05）。结论：TIMM13的表达促进小鼠肝星状细胞的活化和增殖，进而促进了肝纤维化的发展。

       

      Abstract: Objective:To investigate the effect of translocase of inner mitochondrial membrane 13 (TIMM13)expression on the activation and proliferation of mouse hepatic stellate cells and explore the role of TIMM13 in the progression of liver fibrosis.Methods:JS-1 cells were treated with 5 ng/mL, 10 ng/mL 20 ng/mL transform-ing growth factor-β1 (TGF-β1) for 24 h to induce activation of JS-1 cells.The experiment was divided into con-trol group(0 ng/mL group), 5 ng/mL group, 10 ng/mL group and 20 ng/mL group.The mRNA and protein expres-sion levels of TIMM13, fibrosis index α-smooth muscle actin (α-SMA) and collagen I (COL1A1) in each group were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and western blotting.TIMM13 was knocked down and overexpressed in JS-1 cells, and the experiments were divided into empty-vec-tor group (control group) and silence group/overexpression group.The mRNA and protein expression levels of TIMM13, α-SMA and COL1A1 were detected by RT-qPCR and western blotting.The cell counting kit-8 (CCK-8) assay was used to detect the proliferation of TIMM13-silenced/TIMM13-overexpressing cells at 0, 24, 48 and 72 hours.Results:Compared with the control group, the mRNA expression level of TIMM13 in the 5 ng/mL group, 10 ng/mL group and 20 ng/mL group increased (all P< 0.05), and the protein expression of TIMM13 in the 5 ng/mL group and 10 ng/mL group increased(all P< 0.05).Compared with the control group, the mRNA ex-pression levels of TIMM13, α-SMA and COL1A1 in the silencing group decreased (all P< 0.01).The protein expression levels of TIMM13 and α-SMA decreased (all P< 0.01) and the proliferation of JS-1 cells was significantly inhibited at 48 h and 72 h (all P< 0.05) while the mRNA expression levels of TIMM13, α-SMA and COL1A1 increased in the overexpression group(all P< 0.05).The protein expression levels of TIMM13 and α-SMA increased(all P< 0.05)and the prolif-eration of JS-1 cells at 48 h and 72 h was enhanced (all P< 0.05).Conclusion:The expression of TIMM13 pro-motes the activation and proliferation of hepatic stellate cells in mice, thereby facilitating the development of liv-er fibrosis.

       

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