摘要目的 观察重组转化生长因子β1(TGFβ1)和β3(TGFB3)基因对大鼠肝星状细胞株(HSC-T6)的基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)及金属蛋白酶组织抑制剂-1(TIMP-1)表达的影响.方法 (1)构建质粒pcDNA3.1(+)-TGFB,和peDNA3.1(+)-TGFβ3;(2)瞬时转染:将pcDNA3.1(+)-TGFβ1和peDNA3.1(+)-TGFβ3分别及共同转染HSC-T6细胞,荧光定量PCR法和免疫印迹法检测转染后24、48和72 h时MMP-2、MMP-9、TIMP-1 mRNA和蛋白的表达;(3)稳定转染:pcDNA3.1(+)-TGFβ1转染HSC-T6细胞,经筛选建立高表达TGFβ1的HSC-T6细胞克隆,pcDNA3.1(+)-TIGFβ3转染细胞克隆,48 h后检测MMP-2、MMP-9和T1MP-1的表达.结果 (1)TGFβ1转染细胞后,MMP-2和TIMP-1明显增高(P＜0.05),MMP-9无变化;TGFβ3转染细胞后,MMP-2和MMP-9无变化,TIMP-1 mRNA也无变化,但TIMP-1蛋白明显增高(P＜0.05);共转染组的MMP-2较空白组和对照组明显增加(P＜0.05),MMP-9无改变,TIMP-1较TGFβ1转染组明显下降(P＜0.05).(2)TGFβ3转染克隆细胞后,MMP-2无明显变化,MMP-9明显增加(P＜0.05),TIMP-1明显下降(P＜0.05).结论 重组TGFβ3基因可能通过促进MMP-2或MMP-9的活性和抑制TIMP-l的活性而发挥抗肝纤维化作用.

abstractsObjective To investigate the effects of transforming growth factorβ1(TGFβ1)and β3 (TGFβ3)gene transfer on MMP-2,MMP-9 and TIMP-1 expression in hepatic stellate cells(HSC-T6).Methods TGFβ1 and TGFβ3 expression plagmids were constructed.The recombinant expression plasmid pcDNA3.1(+)-=TGFβ1 and pcDNA3.1(+).TGFβ3 were transfected or cotransfected into HSC-T6.At 24,48 and 72 h after transfection,the expression of MMP-2,MMP-9 and TIMP-1 mRNA were detected by real-time quantitative PCR,and the expression of MMP-2,MMP-9 and TIMP-1 protein were detected by Western blot.The recombinant expression plasmid pcDNA3.1(+).TGFβ1 was transfected into HSC-T6,and positive clones were selected by G418.The positive clones were transfected by the recombinant expression plasmid pcDNA3.1(+).TGFβ1,and the expression of MMP-2,MMP-9 and TIMP-1 were detected at 48 h after transfection.Results After transfection with peDNA3.1-TGFβ1,MMP-2 and TIMP-1 increaged remarkably in HSC-T6 cells(P＜0.05),but MMP-9 remained at the sanle level;After transfection with pcDNA3.1-TGFβ3,expression levels of MMP-2,MMP-9 and TIMP-1 mRNA were not changed,but TIMP-1 protein increased remarkably(P＜0.05);in cotransfection group,the expression of MMP-2 was higher than that in the blank and the control groups(P＜0.05),but MMP-9 level was not changed and TIMP-1was decreased compared with that in the TGF-β1 transfection group(P＜0.05).After TGFβ3was transfected into positive clones,the change of MMP-2 wag not significant(P＞0.05).but MMP-9 increaged and TIMP-1 decreased significantly at 48 h after transfection(P＜0.05).Conclusions TGFB3 may inhibit liver fibrosis by increase the activity of MMP-2 and MMP-9,and decrease the activity of TIMP-1.