重组转化生长因子β3基因对大鼠肝星状细胞Ⅰ型胶原表达的影响

目的观察转化生长因子D3基因（TGFβ3）对大鼠肝星状细胞株（HSC—T6）Ⅰ型胶原合成的影响。方法TGFβ3表达质粒pcDNA3．1（＋）-TGFβ31和TGFβ1表达质粒pcDNA3．1（＋）-TGFD11的构建。通过脂质体介导方法，将pcDNA3．1（＋）-TGFβ1、pcDNA3．1（＋）-TGFβ3分别及共同转染体外培养的HSC—T6细胞，荧光定量PCR法及Westernblot法分别检测转染后TGFβ1、TGFD3、Ⅰ型胶原mRNA及蛋白质的表达。将pcDNA3．1（＋）-TGFD1转染HSC—T6细胞，经G418筛选建立高表达TGFD1的HSC～T6细胞克隆，pcDNA3．1（＋）-TGFD3转染克隆细胞，荧光定量PCR法检测转染后TGFβ3、TGFβ1及Ⅰ型胶原mRNA的表达，Westernblot法检测TGFβ1、Ⅰ型胶原蛋白的表达情况。结果构建的pcDNA3．1（＋）TGFD3、pcDNA3．1（＋）-TGFD1质粒可转染HSC—T6细胞，转染率28．2％。pcDNA3．1（＋）TGF侈3转染细胞后，Ⅰ型胶原mRNA及蛋白的表达较空白组及对照组增加，以72h增高最为明显（P〈0．05）；共转染组Ⅰ型胶原mRNA及蛋白质的表达较pcDNA3．1（＋）-TGFβ1转染组明显降低（P〈0．05）。TGF侈3转染克隆细胞后，TGFD1mRNA表达较克隆组无明显改变（P〉0．05），而蛋白质表达明显下降（P〈0．05），Ⅰ型胶原mRNA及蛋白质表达均较克隆组明显降低（P〈0．05）。结论TGFD3基因转染正常培养的HSC—T6细胞，增加Ⅰ型胶原的表达；转染高表达TGFβ1的克隆组HSC—T6细胞，Ⅰ型胶原表达明显降低，提示TGFβ3对肝纤维化的发生有抑制作用。

Effects of transforming growth factor-beta 3 gene transfer on type I collagen synthesis of hepatic stellate cells

OBJECTIVE: To observe the effects of transforming growth factor-beta 3 gene transfer on type I collagen synthesis of cells of HSC-T6. METHODS: Transforming growth factor-beta 1 expression plasmid and transforming growth factor-beta 3 expression plasmid were constructed. The recombinant expression plasmids pcDNA3.1(+)-TGF beta 1 and pcDNA3.1(+)-TGF beta 3 were respectively transfected and cotransfected into cultured HSC-T6 cells; expression of TGF betav1, TGF beta 3, type I collagen mRNA were detected by real-time quantitative PCR, expression of TGF beta 1 and type I collagen protein were detected by Western blot. The recombinant expression plasmid pcDNA3.1(+)-TGF beta 1 was transfected into cultured HSC-T6 cells; positive clones were selected by G418. The positive clones were transfected by the recombinant expression plasmid pcDNA3.1(+)-TGF beta 3; expression of TGF beta 1, TGF beta 3 and type I collagen mRNA were detected by real-time quantitative PCR; expression of TGF beta 1 and type I collagen protein were detected by Western blot. RESULTS: HSC-T6 was transfected by recombinant expression plasmid pcDNA3.1(+)-TGF beta 1 and pcDNA3.1(+)-TGF beta 3 and the transfection efficiency was 28.2%. After the cells were transfected with pcDNA3.1-TGF beta 3, type I collagen mRNA and the protein expression in the cells were higher than those in the untransfected cells (control group) (P < 0.05). The increase reached to the maximal at 72 h after the transfection. Expressions of type I collagen mRNA and the protein in the cells transfected by pcDNA3.1(+)-TGF beta 1 were higher than in those cotransfected by pcDNA3.1(+)-TGF beta 1 and pcDNA3.1(+)-TGF beta 3 (P < 0.05). TGF beta 1 protein, type I collagen mRNA and type I collagen protein expression significantly decreased in the clones transfected by recombinant expression plasmid pcDNA3.1(+)-TGF beta 3 (P < 0.05), but the changes of TGF beta 1 mRNA were not significant (P > 0.05). CONCLUSIONS: Expression of type I collagen increased after the cultured HSC-T6 cells were transfected by TGF beta 3 gene. The significant decrease of the expression of type I collagen of the TGF beta 3 gene transfected positive clones suggests that TGF beta 3 could inhibit the occurrence of liver fibrosis.