结缔组织生长因子诱导大鼠前体细胞(WB-F344)向肝脏实质细胞分化

目的观察结缔组织生长因子（connective tissue growth factor,CTGF）对大鼠肝脏前体细胞（WB—F344）分化的影响，进一步观察细胞外基质的变化。方法采用MTF法检测CTGF对WB—F344细胞活性的影响。采用Wester Rblotting方法分别检测CTGF诱导WB—F344细胞后胆管细胞标志物CK-19及肝细胞标记物AFP、ALB的变化。采用Western blotting和实时荧光定量PCR检测细胞外基质相关指标金属蛋白酶组织抑制剂（tissue inhibitor of metalloproteinase．TIMP）-1在mRNA水平和蛋白水平的变化。结果作用于WB—F344细胞24h后，CTGF组的细胞活性大于转化生长因子-β组（P〈0．05）。5ng／ml CTGF作用于WB—F344细胞24h后，可以明显改变WB—F344细胞表面标记物在蛋白水平的表达。幼稚肝细胞标记物AFP的表达是对照组的0．38倍（P=10．002），成熟肝细胞标记物ALB和胆管细胞标记物CK-19的表达均增加，分别为对照组的3．5倍和1．58倍（P分别为0．000、0．002）。同时，5ng／ml CTGF可以上调WB—F344细胞TIMP-1在mRNA水平和蛋白水平的表达，分别为对照组的2．47倍和3．36倍（P=0．000、0．002）。结论5ng／ml CTGF有诱导肝脏前体细胞向肝脏实质细胞分化的趋势，并伴有WB—F344细胞外基质相关指标TIMP-1表达的上调。

Differentiation of rat hepatic progenitor cells(WB-F344 cells) into hepatic parenchymal cells induced by connective tissue growth factor

Objective To observe the influence of connective tissue growth factor （CTGF） on the differentiation of rat hepatic progenitor cells （WB-F344 cells）, and the changes of extracellular matrix during this procedure. Methods MTT assay was used to evaluate the influence of CTGF on the viability of WB-F344 cells. Western blotting was used to detect the expressions of cytokeratin 19 （CK- 19 ）, α-fetoprotein （AFP） and albumin （ALB） after WB-F344 cells were induced by CTGF. Western blotting and real-time fluorescent quantitative PCR were used to detect mRNA and protein expressions of tissue inhibitor of metalloproteinase （TIMP）-1. Results Activity of WB-F344 cells stimulated by CTGF for 24 hours was higher than that stimulated by transforming growth factor-β （P〈0.05）. After WB-F344 cells had been stimulated by CTGF at the concentration of 5 ng/ml for 24 hours, the protein expressions of the phenotype markers of WB-F344 cells changed significantly. The expression of AFP, the fetal hepatocyte marker, was 3.8 times that of the control group （P=0.002）, while the expressions of ALB, the mature hepatocyte marker, and CK- 19, the cholangiocyte marker, 3.5 and 1.58 times those of the control group （P=0.000, 0.002）. Meanwhile, the expressions of TIMP-1 both in mRNA level and protein level were up-regulated when WB-F344 cells were stimulated by CTGF at the concentration of 5 ng/ml, 2.47 and 3.36 times compared with the control group, respectively （P=0.000, 0.002）. Conclusions CTGF at the concentration of 5 ng/ml can induce the differentiation of WB-F344 cells into hepatic parenchymal cells, while the expression of TIMP-1 is found to be up-regulated.