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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 investigate the effects of recombinant transforming growth factor-β3 (TGF-β3) on the synthesis and secretion of protein in and out of rat hepatic stellate cells (HSCs). Methods Plasmid pcDNA3.1 (+) - TGF-β3 and pcDNA3.1 (+) - TGF-β1 were constructed. Stable transfection: pcDNA3.1 (+) - TGF-β1 was transfected into HSC-T6 by liposome-mediated method and then screened by G418 to establish a stable and stable expression vector pcDNA3.1 (+) - TGF- T6 cell positive clone. The HSC-T6 cells were transfected with pcDNA3.1 (+) - TGF-β3. The expression of TGF-β1, type Ⅰ collagen and matrix metalloproteinase (MMP-2) were detected by Western blotting and enzyme-linked immunosorbent assay (ELISA) (MMP) -2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP) -1 protein expression. Results After HSC-T6 positive cells were transfected with pcDNA3.1 (+) - TGF-β3, the protein expressions of TGF-β1, type Ⅰ collagen and TIMP-1 were significantly higher than those of the positive clones (P <0.05). The expression of MMP-2 also decreased, but there was no significant difference between the two groups (P> 0.05), while the expression of MMP-9 was significantly increased (P <0.05). Conclusion The recombinant plasmid pcDNA3.1 (+) - TGF-β3 can reduce the synthesis and secretion of type Ⅰ collagen in and outside cells. By regulating the expression of MMP-9 and TIMP-1, the synthesis of extracellular matrix can be inhibited and the degradation can be accelerated.