摘要
目的探讨肝细胞生长因子（HGF）与表皮细胞生长因子（EGF）联合诱导大鼠骨髓间充质干细胞（MSCs）分化为类肝细胞的可行性。方法取大鼠股骨骨髓，用直接贴壁法分离纯化Mats并体外传代，实验组用HGF（20mg／m1）＋EGF（10mg／m1）对体外培养的Mscs进行诱导分化。细胞免疫化学法及流式细胞仪对培养的MSCS进行鉴定。RT-PCR检测诱导后慨的AFP、AIb、CK-18的mRNA表达。电镜观察诱导细胞的超微结构。结果贴壁的MSCs单个存在或形成克隆，细胞形态比较均一，为长梭形，7～10天达汇合。免疫细胞化学及流式细胞仪均证明培养的细胞为MSCs。实验组MSCS在培养21天时表现为类肝细胞的形态特点。RT-PCR：AFP mRNA在第7天呈阳性表达，AIb mRNA及CK-18 mRNA开始即有表达，21天增强。电镜观察见诱导21天的Mats形态结构与肝细胞相吻合。结论Mats在体外容易分离培养和扩增，HGF＋EGF可诱导MSCs向类肝细胞分化，为细胞移植治疗肝衰竭提供新的探索思路。
Objective To research the possibility of the rat mesenchymal stem cells （MSCs） to differentiate into hepatocyte-like with hepatocyte growth factor plus E growth factor in vitro. Methods （1） Bone marrow in the femurs of Wistar rat was collected by flushing under sterile condition. MSCs were separated and cultured according to the direct anchoring method, identified by using immunocytochemi- cal methods and flow cytometry. After MSCs were induced by HGF （20mg/ml） and EGF （10mg/ml）, mRNA expression level of AFP. AIb.CK-18 in the MSCs were detected by RT-PCR In addition, ultrastructures in the cells induced were observed using electron microscope. Results Anchored MSCs is single or cell clone is developed. Cell is uniform in the configuration and converged long fusiform after 7-10 days. Cultured cells are certified to be MSCs in the immunocytochemistry and flow cytometry. Afer 21 days, cultured cells show Hepatocyte-like characters in the configuration. RT-PCR：On day 7 mRNA of AFP was detected; mRNA of Alb and CK-18 were detected at the beginning, then strengthen on 21 day. At the same time, induced cells in the electron microscope results coincided with hepatic cells. Conclusions MSCs can be isolated, cultured and expanded easily in vitro. MSCs induced by HGF plus EGF in vitro can differentiate into hepatocyte-like cells. As a result, it can provide a new therapeutic method for liver failure.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2006年第5期446-449,共4页
Medical Journal of Chinese People's Liberation Army
基金
广东省自然科学基金资助项目(2001-010593
2002-010593)
