论文部分内容阅读
目的 明确多种生长因子对胚胎肝脏前体细胞体外增殖分化的影响。方法 用胶原酶从胎龄14.5d SD大鼠胚胎肝脏分离单个细胞,采用~3H-TdR掺入法检测生长因子对胎肝细胞体外增殖的促进作用,并观察生长因子及细胞外基质成分对胎肝干细胞集落形成的影响。采用免疫细胞双标记及G-6-P酶活性测定以检测肝前体细胞表面标记的表达及向成熟肝细胞的分化能力。结果 肝前体细胞在体外培养时显示克隆样生长的特性。促肝细胞生长因子(HGF)、表皮生长因子(EGF)能促进肝前体细胞的增殖,使DNA合成加速,并促进干细胞集落的形成及角蛋白19、白蛋白、G-6-P的表达。转化生长因子α对肝前体细胞的促增殖作用较弱。转化生长因子β对肝前体细胞的增殖起到抑制作用。细胞基质成分Ⅰ型胶原、Ⅳ型胶原、层黏连蛋白能促进肝干细胞集落的形成,而纤维连接蛋白的作用较弱。肝干细胞单克隆增殖需要生长因子及细胞外基质的共同参与,加入新鲜分离胎肝细胞培养液上清液时单细胞增殖较快,于第5天即形成细胞集落。结论 HGF、EGF对肝前体细胞的增殖分化起重要作用,细胞外基质成分亦参与了其增殖分化过程。肝干细胞单克隆培养除需生长因子和细胞外基质外,可能亦有某些造血细胞、间质细胞分泌的因子参与其增殖分化的调控。
Objective To investigate the effect of multiple growth factors on proliferation and differentiation of embryonic liver precursor cells in vitro. Methods Collagenase was used to separate single cells from embryonic liver of embryonic 14.5 d SD rats. 3H-TdR incorporation was used to detect the effect of growth factors on the proliferation of fetal hepatocytes in vitro. Growth factors and extracellular matrix components Effect of Hepatic Stem Cell Colony Formation. Immunocytochemical double labeling and G-6-P enzyme activity assay were used to detect the expression of surface markers of liver precursor cells and the differentiation ability to mature hepatocytes. Results Liver precursor cells showed clonally-like growth characteristics when cultured in vitro. Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) can promote the proliferation of hepatic precursor cells, accelerate DNA synthesis and promote the formation of stem cell colonies and the expression of keratin 19, albumin and G-6-P . Transforming growth factor-alpha has a weak effect on promoting proliferation of liver precursor cells. Transforming growth factor beta inhibits the proliferation of hepatic precursor cells. Collagen type I collagen, type IV collagen, laminin can promote the formation of hepatic stem cell colonies, and the role of fibronectin is weak. Monocyte proliferation of hepatic stem cells requires the participation of both growth factors and extracellular matrix. When fresh supernatant of fetal hepatocytes is added, single cell proliferation is faster, and colony formation occurs on the fifth day. Conclusion HGF and EGF play an important role in the proliferation and differentiation of hepatic precursor cells. Extracellular matrix components are also involved in the process of proliferation and differentiation. In addition to the growth factors and extracellular matrix, there may also be some hematopoietic cells in the monoclonal culture of hepatic stem cells, and the factors secreted by interstitial cells are involved in the regulation of proliferation and differentiation.