TGF-β1信号介导的肝星状细胞促进胚胎肝前体细胞向胆管细胞方向分化

TGF-β1 signaling mediated hepatic stellate cells promote hepatic progenitor cells differentiation into cholangiocytes

目的探讨转化生长因子β1(transforming growth factorβ1,TGF-β1)激活的小鼠肝星状细胞(mouse hepatic stellate cells,mHSCs)对小鼠胚胎肝前体细胞(mouse hepatic progenitor cells,mHPCs)分化的影响。方法 mHSCs细胞株加入10 ng/mL TGF-β1刺激48 h,并分为激活组(mHSCsTGF-β1)和对照组(mHSCs)。qRT-PCR和Western blot法检测mHSCs激活情况。采用荧光激活细胞筛选法(fluorescence-activated cell sorter,FACS)分选DLK1(delta-like1 homologue)表面抗原阳性的原代mHPCs,分选的细胞利用免疫荧光染色法观察甲胎蛋白(alpha-fetoprotein,AFP)、白蛋白(albumin,ALB)及细胞角蛋白19(cytokeratin 19,CK19)抗原表达情况。mHPCs与mHSCs Transwell共培养6 d后,分为mHPCs+mHSCs-TGF-β1组、mHPCs+mHSCs组和mHPCs组。细胞免疫荧光技术法和qRTPCR法检测分化标志物表达情况。结果激活组mHSCs中TGF-β1、α-SMA、TGF-β-R1、Jagged1、VEGF、HGF等m RNA和TGF-β1、a-SMA、TGF-β-R1、Jagged1等蛋白的表达量均较对照组明显增加(P<0.01);FACS新分选的mHPCs胞质中表达AFP和少量ALB,但不表达CK19;mHPCs+mHSCs-TGF-β1组mHPCs胞质中大量表达胆管细胞标志物,并且CK19、SOX9、Hes1等m RNA表达量均较其他两组明显增加,而其他两组mHPCs中ALB、AFP等的表达量明显增加(P<0.01)。结论 TGF-β1激活的肝星状细胞诱导胚胎肝前体细胞向胆管细胞分化。

Objective To investigate the effects of mouse hepatic stellate cells （mHSCs） activated by transcription factor β1 （TGF-β1） on mouse hepatic progenitor cells （mHPCs） differentiation. Methods After the mHSCs were treated with 10 ng/mL TGF-β1 for 48 h, the cells were assigned into activation group （mHSCs-TGF-β1）, and those without treatment served as control group （mHSCs）. Activation of mHSCs was detected by qRTPCR and Western blotting. mHPCs were isolated from fetal livers based on the cell surface antigen deltalike protein 1 （Dlk1） by a fluorescence-activated cell sorter （FACS）, and the expression of alpha-fetoprotein （AFP）, albumin （ALB）, and cytokeratin 19 （CK19） was observed by immunocytochemical assay. Then the obtained mHPCs and mHSCs were co-cultured in a Transwell system for 6 d. Immunocytochemical assay and qRTPCR were used to detect the expression of differentiation markers in mHPCs＋mHSCs-TGF-β1 group, mHPCs＋mHSCs group and mHPCs group. Results The mRNA levels of a-SMA, TGF-β1, TGF-β-R1, Jagged1, HGF and VEGF, and protein levels of TGF-β1, a-SMA, TGF-β-R1 and Jagged1 were significantly higher in the activation group （P〈0.01）; Freshly sorted mHPCs by FACS expressed AFP and low level of ALB but not CK19 in the cytoplasm. In the mHPCs＋mHSCs-TGF-β1 group, mHPCs highly expressed ductspecific markers in the cytoplasm, and the mRNA levels of CK19, SOX9 and Hes1 were obviously higher than the other 2 groups of cells. But the ALB and AFP expression was more obvious in the 2 groups （P〈0.01）. Conclusion TGF-β1-activated hepatic stellate cells induce hepatic progenitor cells differentiation into cholangiocytes.