Gao C, Gressner G, Zoremba M, Gressner A M
Department of Clinical Chemistry, Philipps University, Marburg, Germany.
J Cell Physiol. 1996 Jun;167(3):394-405. doi: 10.1002/(SICI)1097-4652(199606)167:3<394::AID-JCP3>3.0.CO;2-K.
It is still a subject of debate whether hepatocytes have the ability to express TGF-beta. Therefore, we investigated in freshly isolated and in monolayer cultures of rat hepatocytes the expression of TGF-beta isoform s at the RNA and protein level applying RT-PCR, immunocytochemistry, immunoblotting, and functional assays of TGF-beta. TFG-beta 1, -beta 2, and -beta 3 transcripts were detected in cultured cells, and the level of m RNA increased up to 48/72 h, but TGF-beta 1 transcripts were absent in freshly isolated cells. Using APAAP stainings the proteins of all three TGF-beta isoforms were observed in hepatocyte cultures from 5-96 h, but in hepatocytes in the liver in situ and in freshly isolated cell suspensions TGF-beta staining was negative. SDS-PAGE under reducing conditions followed by Western blotting detected in cell lysates the subunit of mature TGF-beta at about 13 kd. Analysis of TGF-beta bioactivity with the mink cell (Mv1Lu) proliferation inhibition assay and competitive radioligand assay confirmed in activated (i.e., acidified and subsequently neutralized) hepatocyte-conditioned media the presence of TGF-beta, which, however, is almost entirely in the latent form. It is concluded that TGF-beta can be expressed in cultured hepatocytes and that the level of expression is quickly upregulated under abnormal, not yet known, microenvironmental conditions.
肝细胞是否具有表达转化生长因子-β（TGF-β）的能力仍是一个有争议的话题。因此，我们运用逆转录聚合酶链反应（RT-PCR）、免疫细胞化学、免疫印迹以及TGF-β的功能测定，在新鲜分离的大鼠肝细胞和单层培养的肝细胞中，研究了TGF-β异构体在RNA和蛋白质水平的表达情况。在培养的细胞中检测到了TGF-β1、-β2和-β3转录本，mRNA水平在48/72小时内升高，但在新鲜分离的细胞中未检测到TGF-β1转录本。使用碱性磷酸酶-抗碱性磷酸酶（APAAP）染色法，在5至96小时的肝细胞培养物中观察到了所有三种TGF-β异构体的蛋白质，但在原位肝脏中的肝细胞以及新鲜分离的细胞悬液中，TGF-β染色呈阴性。在还原条件下进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），随后进行蛋白质免疫印迹分析，在细胞裂解物中检测到了约13千道尔顿的成熟TGF-β亚基。用貂肺上皮细胞（Mv1Lu）增殖抑制试验和竞争性放射性配体试验分析TGF-β生物活性，证实在活化的（即酸化并随后中和的）肝细胞条件培养基中存在TGF-β，然而，其几乎完全以潜伏形式存在。结论是，TGF-β可在培养的肝细胞中表达，并且在异常但未知的微环境条件下，表达水平会迅速上调。
