HSP47 siRNA对体外培养HTCF细胞生物学行为及TGF-β1表达水平的影响

Effects of HSP47 siRNA on biological behaviors of HTCF cells cultured in vitro and the expression level of TGF-β1

目的:探究热休克蛋白47(HSP47)siRNA对体外培养人眼Tenon囊成纤维(HTCF)细胞生物学行为及转化生长因子-β1(TGF-β1)表达水平影响。方法:体外培养HTCF细胞,并分为:空白对照组、空载体组和转染组;转染组根据HSP47基因序列设计并合成干扰siRNA序列,构建载体并导入HTCF细胞中;空载体组导入空白载体。采用RT-PCR和蛋白质印迹实验检测细胞中HSP47 mRNA和蛋白的表达情况,采用克隆形成实验、流式细胞术、Transwell法及划痕实验检测细胞增殖、凋亡、侵袭及迁移,蛋白质印迹实验检测增殖、凋亡、侵袭、迁移蛋白和TGF-β1的表达情况。结果:相比空载体组,转染组HSP47 mRNA和蛋白的表达、克隆形成率、细胞愈合率、侵袭细胞数目、Ki67、N-cadherin、TGF-β1蛋白相对表达水平显著降低(P<0.05),E-cadherin蛋白相对表达水平显著升高(P<0.05),但细胞凋亡率、Bcl-2和Bax蛋白相对表达水平均无差异(P>0.05)。结论:HSP47 siRNA可以通过抑制TGF-β1蛋白的表达降低HTCF细胞的增殖、侵袭及迁移能力,但对HTCF细胞的凋亡无明显影响。

AIM:To explore the effects of heat shock protein 47(HSP47)siRNA on biological behaviors of human Tenon capsule fibroblasts(HTCF)cells cultured in vitro and the expression level of transforming growth factor-β1(TGF-β1).METHODS:HTCF were cultured in vitro and divided into blank control group,empty vector group and transfection group.In transfection group,interfering siRNA sequences were designed and synthesized based on the HSP47 gene sequences,vectors were constructed and introduced into HTCF.The empty vector group was introduced with empty vectors.The expressions of HSP47 mRNA and protein in cells were detected by RT-PCR and Western blot.The proliferation,apoptosis,invasion and migration of cells were detected by clone formation assay,flow cytometry,Transwell method and scratch test.The expressions of proliferation,apoptosis,invasion and migration proteins,and TGF-β1 were detected by Western blot.RESULTS:Compared with empty vector group,expression of HSP47 mRNA and protein,clone formation rate,cell healing rate,number of invasive cells,relative expression levels of Ki67,N-cadherin and TGF-β1 were significantly decreased in transfection group(P<0.05),relative expression level of E-cadherin protein was significantly increased(P<0.05),but there was no difference in apoptosis rate,and relative expression levels of Bcl-2 and Bax(P>0.05).CONCLUSION:HSP47 siRNA can reduce proliferation,invasion and migration abilities of HTCF cells by inhibiting the expression of TGF-β1 protein,without significant effects on the apoptosis of HTCF cells.