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目的在成功分离并培养大鼠骨髓间充质干细胞的基础上,用HGF、EGF以及HGF+EGF诱导其向肝细胞方向分化,并采用不同方法鉴定细胞分化能力,试图为肝细胞移植等寻找新的种子细胞来源。方法应用HGF、EGF、HGF+EGF诱导大鼠骨髓间充质干细胞分化为肝样细胞。实验分组:A组:未加任何诱导因素的BMSCs;B组:HGF(20ng/ml),诱导分化BMSCs;C组:EGF(10ng/ml),诱导分化BMSCs;D组:HGF(20ng/ml)+EGF(10ng/ml),诱导分化BMSCs。相差显微镜下观察细胞形态的变化;在诱导第7、21天行免疫细胞化学染色检测肝细胞标志AFP和CK18。结果在加入各种诱导成分后,梭形或成纤维细胞样细胞随着诱导时间的延长,变成纺锤形、不规则圆形或多角形细胞,数量逐渐变多,形似肝细胞。在诱导分化第7天和21天免疫细胞化学染色各诱导组均可检测出AFP和CK18表达,图像分析表明HGF+EGF联合诱导分化的AFP、CK18阳性率最高,HGF次之,EGF则较弱。结论 HGF、EGF、HGF+EGF均能诱导大鼠骨髓间充质千细胞分化为肝样细胞,分化后的肝样细胞能分泌肝细胞特异性产物AFP、CK18。
OBJECTIVE: To successfully differentiate rat bone marrow mesenchymal stem cells (MSCs) and to differentiate into hepatocytes with HGF, EGF and HGF + EGF, and to identify cell differentiation ability using different methods in an attempt to find a new cell for hepatocyte transplantation The source of seed cells. Methods HGF, EGF and HGF + EGF induced rat bone marrow mesenchymal stem cells to differentiate into hepatocyte-like cells. Group B: HGF (20ng / ml), induced differentiation of BMSCs; Group C: EGF (10ng / ml), induced differentiation of BMSCs; Group D: HGF (20ng / ml ) + EGF (10 ng / ml) to induce differentiation of BMSCs. The changes of cell morphology were observed under phase contrast microscope. Immunocytochemical staining was used to detect hepatocyte markers AFP and CK18 on the 7th and 21st day after induction. Results After adding various inducing components, the spindle or fibroblast-like cells became spindle-shaped, irregular round or polygonal cells with increasing induction time, gradually increasing in number and forming liver cells. The expression of AFP and CK18 were detected by immunocytochemical staining on the 7th day and the 21st day respectively. The results of image analysis showed that the positive rate of AFP and CK18 induced by HGF + EGF was the highest, followed by HGF, while EGF was weaker . Conclusions HGF, EGF, HGF + EGF can induce rat bone marrow mesenchymal stem cells to differentiate into hepatocyte - like cells. The differentiated hepatocyte - like cells can secrete hepatocyte specific products AFP and CK18.