摘要
目的：寻找临床肝细胞移植和生物型人工肝所需的肝细胞来源，研究肝细胞生长因子（HGF）、表皮生长因子（EGF）和成纤维细胞生长因子（FGF）对人骨髓间充质干细胞（hBMSCs）在体外分化为肝样细胞的影响。方法利用密度梯度离心结合贴壁法分离、纯化培养 hBMSCs，取 P2代对数生长期的 hBMSCs 进行诱导，诱导前将细胞分为4组： A 组加入含 HGF、FGF 的条件培养液；B 组加入含 HGF、EGF 的条件培养液；C 组加入含 HGF、FGF、EGF 的条件培养液；D 组不加任何细胞因子的条件培养液。分别通过逆转录－聚合酶链反应（RT －PCR）和免疫组化法鉴定经诱导后细胞甲胎蛋白（AFP）和白蛋白（ALB）的表达，观察不同诱导条件下 hBMSCs 分化为肝样细胞的能力。结果A、B、C 3组细胞经诱导后均可分化为形态似肝细胞样的细胞，形态无明显不同；免疫组化示 A、B、C 3组细胞均在诱导后7天表达 AFP，21天表达 ALB，以 C 组阳性表达最强；RT －PCR 结果显示 3组细胞经诱导后表达 ALB，并随着时间的延长表达增加；A、B、C 3组细胞在诱导21天时行糖原染色，均可见胞质呈红色，表明诱导后细胞有合成糖原的能力。结论hBMSCs 在体外特定的条件下可定向诱导分化为肝样细胞，尤以EGF、FGF 和 HGF 共同诱导效果最佳。
Objective To search for the source of hepatocytes available for transplantation and bio -artificial liver, and to investigate the effects of HGF, EGF and FGF on the differentiation of human bone marrow mesenchymal stem cells （hBMSCs） into hepatocyte -like cells in vitro.Methods hBMSCs were isolated through gradient centrifugation and cultivated by adherence culture.The cells in logarithmic growth phase were divided into four groups, according to various agents to which they were exposed, namely conditioned media with HGF and FGF （group A）, conditioned media with HGF and EGF （group B）, conditioned media with HGF, FGF and EGF （group C）, and conditioned medium without any cytokines （group D）.Then, the amounts of alphafetoprotein （AFP） and albumin （ALB） were determined by reverse transcription polymerase chain reaction （RT -PCR） and immunohistochemistry methods, in order to assess the capabilities of hBMSCs to differentiate into hepatocyte -like cells under various conditions.Results The cells in groups A, B and C could be induced into hepatocyte -like cells with similar morphology.According to immunohistochemistrical results, the presence of AFP and ALB were determined in groups A, B and C after 7 and 21 days of induction, respectively, with the strongest expression in Group C.RT -PCR showed that, the expression of ALB, which appeared after induction were enhanced as cultivation continued.Furthermore, the cells in groups A, B and C underwent glycogen staining on Day 21 and resulted in visible red stain in the cytoplasm, indicating the capacities of the cells to synthesize glycogen after induction.Conclusions hBMSCs can differentiate into hepatocyte -like cells under certain conditions in vitro, especially when exposed to EGF, FGF and HGF simultaneously.
出处
《徐州医学院学报》
CAS
2015年第3期166-170,共5页
Acta Academiae Medicinae Xuzhou
基金
江苏省高校自然科学研究重大项目
关键词
骨髓间充质干细胞
肝样细胞
诱导分化
bone marrow mesenchymal stem cells
hepatocyte -like cells
induced differentiation
