重组转化生长因子-β_3基因对大鼠肝星状细胞内、外蛋白表达的影响

Effects of recombinant transforming growth factor-β_3 gene on protein expressions intracellular and extracellular of hepatic stellate cell

目的探讨重组转化生长因子-β3(TGF-β3)基因对大鼠肝星状细胞(HSC)内、外蛋白合成及分泌的影响。方法构建质粒pcDNA3.1(+)-TGF-β3和pcDNA3.1(+)-TGF-β1。稳定转染:通过脂质体介导的方法,将pcDNA3.1(+)-TGF-β1转染HSC-T6,经G418筛选建立稳定高表达pcDNA3.1(+)-TGF-β1的HSC-T6细胞阳性克隆。再将pcDNA3.1(+)-TGF-β3转染HSC-T6克隆细胞,用W estern b lot法和酶联免疫吸附法(ELISA)分别检测细胞内外TGF-β1、Ⅰ型胶原、基质金属蛋白酶(MMP)-2、MMP-9及金属蛋白酶组织抑制剂(TIMP)-1的蛋白表达量。结果pcDNA3.1(+)-TGF-β3转染HSC-T6阳性细胞克隆后,与单纯阳性克隆组相比,TGF-β1、Ⅰ型胶原、TIMP-1细胞内蛋白表达及培养上清中的分泌水平均明显降低(P<0.05),MMP-2的表达也降低,但两者间差异无统计学意义(P>0.05),而MMP-9的表达明显增高(P<0.05)。结论重组质粒pcDNA3.1(+)-TGF-β3能减少细胞内外Ⅰ型胶原的合成及分泌;通过调节MMP-9及TIMP-1的表达,可抑制细胞外基质的合成,促进其降解。

Objective To explore the influence of recombinant transforming growth factor-β3（TGF-β3） on protein synthesis and secretion intracellular and extracellular of hepatic stellate cell（HSC）.Methods Plasmid pcDNA3.1（＋）-TGF-β3 and pcDNA3.1（＋）-TGF-β1 were well constructed.A positive cell clone stably and highly expressing TGF-β1 was established after being screened by G418 medium,and the positive clones were transfected by recombinant TGF-β3.HSC-T6 cells were cultured and divided into four groups： blank control group;pcDNA3.1（＋）-enhanced green fluorescent protein（EGFP）-transfected group（negative control group）;pcDNA3.1（＋）-TGF-β1 transfected group（TGF-β1 positive clone group）;pcDNA3.1（＋）-TGF-β3 transfected positive clones group（TGF-β3 interfered group）.Western blot and ELISA were respectively used to detect the protein synthesis and secretion of TGF-β1,typeⅠ collagen,matrix metalloproteinase（MMP）-2,MMP-9 and tissue inhibitor of metalloproteinase（TIMP）-1.Results The protein expressions of TGF-β1,type Ⅰ collagen,MMP-2 and TIMP-1 in the TGF-β1 positive clone group were obviously higher than those in the blank control group and negative control group（P〈0.05）,while the protein expression of MMP-9 was significantly lower.Compared with the TGF-β1 positive clone group,the protein expressions of TGF-β1,type Ⅰcollagen and TIMP-1 in TGF-β3 interfered group were much lower（P〈0.05）,and MMP-2 was lower,but the difference had no statistical significance.The protein expression of MMP-9 was obviously higher（P〈0.05）.Conclusion Recombinant TGF-β3 reduces the synthesis and secretion of type Ⅰ collagen,and inhibits the collagen deposition by adjusting the expression of matrix metalloproteinases and their inhibitors.