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- 2018
转染DNMT1 siRNA对瘢痕疙瘩成纤维细胞生长的影响
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Abstract:
目的:探讨以siRNA干扰DNA甲基转移酶1(DNMT1)表达对瘢痕疙瘩成纤维细胞生长的影响及其机制。方法:采用qRT-PCR和Western blot检测体外培养的正常皮肤细胞和瘢痕疙瘩成纤维细胞中DNMT1 mRNA和蛋白的表达情况。培养瘢痕疙瘩成纤维细胞,分别转染阴性对照siRNA和DNMT1 siRNA,以不做任何处理的细胞为对照,MTT法检测各组细胞的增殖情况,流式细胞仪检测细胞的凋亡率,Western blot 检测细胞中DNMT1蛋白的表达,qRT-PCR检测细胞中DNMT1、PCNA、Bcl-2、Bax和TGF-β1 mRNA的表达情况。结果:DNMT1 mRNA和蛋白在瘢痕疙瘩成纤维细胞中的表达水平较正常皮肤细胞升高(P<0.05)。转染DNMT1 siRNA后细胞的增殖能力减弱,凋亡增强,且DNMT1蛋白和DNMT1、PCNA、Bcl-2和TGF-β1 mRNA表达水平均降低,而Bax mRNA表达水平升高(P<0.05)。结论:DNMT1在瘢痕疙瘩成纤维细胞中高表达,干扰其表达能够明显抑制细胞的增殖,促进细胞凋亡,其作用机制可能与上调Bax和下调PCNA、Bcl-2、TGF-β1表达有关。
Aim:To investigate the effect of RNA interference DNMT1 expression on the growth of keloid fibroblasts and its mechanism.Methods:The expressions of DNMT1 mRNA and protein in normal skin cells and keloid fibroblasts in vitro were detected by qRT-PCR and Western blot.Cultured keloid fibroblasts were transfected with negative control siRNA and DNMT1 siRNA respectively,and the cells without any treatment were used as the control group.The proliferation of cells was tested by MTT method, and the apoptosis of cells was examined by flow cytometry. The expression of DNMT1 protein in the cells of each group was tested by Western blot, and the expressions of DNMT1, PCNA, Bcl-2, Bax and TGF-β1 mRNA in each group were measured by qRT-PCR.Results:The expression levels of DNMT1 mRNA and protein in keloid fibroblasts were significantly higher than those of normal skin cells(P<0.05).After transfection of DNMT1 siRNA, the proliferation of cells was weakened and apoptosis was enhanced, and the relative expression levels of DNMT1 protein and DNMT1, PCNA, Bcl-2 and TGF-β1 mRNA were significantly decreased, while the expression level of Bax mRNA increased significantly(P<0.05).Conclusion:DNMT1 is highly expressed in keloid fibroblasts, interfering with its expression can significantly inhibit cell proliferation and promote cell apoptosis,which may be related with up-regulation of Bax and down-regulation of PCNA, Bcl-2 and TGF-β1 expression
