纤维蛋白凝胶复合bFGF对胚胎间充质干细胞增殖和ALP活性的影响

目的探讨大鼠胚胎间充质干细胞在纤维蛋白凝胶(FG)与碱性成纤维细胞生长因子(bFGF)复合培养后对细胞增殖和碱性磷酸酶(ALP)活性的作用,为将FG应用于组织工程奠定基础。方法实验分为4组:FG组,bFGF组,FG+bFGF组和对照组(采用无凝胶正常培养基培养)。无菌条件下分离培养大鼠胎肢细胞,采用组织块消化法获取胚胎间充质干细胞,取传至第3代细胞接种于以上培养基中,在不同时间点通过倒置相差显微镜观察细胞形态学变化,荧光分光光度计测定细胞增殖指数,酶标仪分析细胞ALP活性,RTPCR检测ALP mRNA表达。结果 FG+bFGF组细胞培养至14d,细胞具有较长突起且连接成网,而对照组细胞呈纺锤形或立方形。各组细胞荧光强度随培养时间的延长逐渐增强,FG+bFGF组在21d时达到最高。FG+bFGF组培养7d、14d、21d ALP活性和ALP mRNA表达均较FG组或bFGF培养组高(0.05)。结论纤维蛋白凝胶复合bFGF可促进大鼠胚胎间充质干细胞增殖,明显增强碱性磷酸酶活性。

Effects of fibrin gels combined with bFGF on proliferation and alkaline phosphatase activity of embryonic mesenchymal stem cells in rat

Objective To study the effects of fibrin gels combined with basic fibroblast growth factor（bFGF） on proliferation and alkaline phosphatase activity of embryonic mesenchymal stem cells in rat to provide a basis for use of fibrin gel（FG） in tissue engneering. Methods Four groups were included： FG group, bFGF group, FG＋bFGF group and normal control group（cells seeded in normal culture medium）. Rat embryonic mesenchymal stem cells were isolated and cultured under aseptic condition using tissue digesting method, the third generation cells were used for each group. The cell morphology was observed by inverted phase contrast microscope, cell proliferation was assessed by fluorospect- rophotometer. Alkaline phosphatase（ALP） activity was detected by automatic biochemistry analysis, and RT-PCR was used to analyze ALP mRNA expression. Results The cultured cells were connected to each other to form network with long processes in FG＋bFGF group, but not to form network with fusiform or cube shape in control group at 14 days. The fluorescence intensity was increased gradually with time in all groups, the highest in FG＋bFGF group than in other groups at 21 days （P〈 0.05）. ALP activity and mRNA expression level were higher in FG＋bFGF group than in other groups （p〈 0.05） at 7, 14 and 21 days. Conclusion Fibrin gels combined with bFGF increased ALP activity and promoted proliferation of rat embryonic mesenchymal stem cells.