不同血清浓度对bFGF联合EGF诱导BMSCs向神经细胞分化的影响

Influence of different serum concentrations on inducing BMSCs into neural cells by epidermal growth factor and basic fibroblast growth factor

目的:探讨不同血清浓度条件下碱性成纤维生长因子(basic fibroblast growth factor,bFGF)联合表皮生长因子(epidermal growth factor,EGF)诱导骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)向神经细胞分化方向的影响。方法:全骨髓贴壁法体外分离培养BMSCs,流式细胞法检测BMSCs表面骨髓基质标志CD90和造血细胞标志CD45,MTT法绘制第3(P3)代BMSCs生长曲线。取P4代细胞分为3组给予不同诱导条件,分别给予含20 ng/ml的bFGF和20 ng/ml EGF的无胎牛血清(A组)、10 ml/L胎牛血清(B组)及100 ml/L胎牛血清(C组)的DMEM/F-12培养液进行诱导,倒置显微镜观察细胞的形态变化,诱导6 d时免疫组织化学法检测细胞内神经元特异性烯醇化酶(NSE)及胶质纤维酸性蛋白(GFAP)的表达,台盼蓝染色检测各组细胞的活力。结果:BM-SCs经3-4次传代培养后形态大致呈单一的长梭形或扁平形,流式细胞鉴定显示CD90阳性,CD45阴性;细胞生长曲线近似呈"S"形;诱导后可见细胞胞体收缩,呈双极、多极并伸出突起;诱导6 d时3组活细胞率分别为:86.57%、95.29%、97.32%;免疫组化各组均见NSE、GFAP表达,3组NSE阳性率高于GFAP,A组NSE阳性率高于B、C组,差异具有统计学意义(P<0.01),C组NSE阳性率高于B组,差异具有统计学意义(P<0.01),A组GFAP阳性率低于B、C组,差异具有统计学意义(P<0.01),C组GFAP阳性率高于B组,差异具有统计学意义(P<0.01)。结论:bFGF联合EGF可诱导BMSCs向神经细胞分化,在无血清条件下趋向于向神经元分化,在高浓度血清条件下趋向胶质细胞分化。

Objective： To study the influence of different serum concentrations on inducing bone mesenchymal stem cells （BMSCs） into neural cells by epidermal growth factor （EGF） associate with basic fibroblast growth factor （bFGF） in vitro. Methods：BMSCs were harvested by the whole bone marrow adherence method in vitro, assay the marrow stromal surface marker CDgo and hematopoietic cell marker CD45 by flow cytometry and draw cell growth curve of P3 generation by MTT assay. BMSCs at passage 4 were divided into 3 groups according different induce conditions, DMEM/F-12 culture mediums which contain 20 ng/ml bFGF, 20 ng/ml EGF and no fetal bovine serum （group A） , 10 mg/L fetal bovine ser- um （group B） and 100 mg/L fetal bovine serum （group C ） were given to induce respectively. Then the cellular morpho-logic change was observed under inverted phase contrast microscope. Immunohistochemical staining was used to detect the expressions of neuron specific enolase （NSE） and collagen fibre acidic protein （GFAP） and Trypan blue staining was used to detect cellular viability. Results： BMSCs roughly present a single long spindle or flat-shaped morphology after 3-4 times subcultured. The flow cytometry identification showed CDgo positive and CD45 negative; the cell growth curve was similar to the ＂S＂. After induction, BMSCs become shrinkage, display bipolar, muhipolar and extend processes. Living cells rates of each group were 86.57%, 95.29%, 97.32%, respectively. Immunohistochemical staining showed that NSE- and GFAP-immunoreactivities were observed in each group, NSE positive rate was higher than GFAP in all groups, and NSE positive rate in group A was significantly higher than those in group B and C （P 〈0. O1 ）, NSE positive rate in group C was significantly higher than that in group B （P 〈0.01 ） ; GFAP positive rate in group A was significantly lower than those in group B and C （P 〈0. 01 ）, but GFAP positive rate in group C was significantly higher than that in group B （P 〈 0. 01 ）. Conelusion：bFGF associate with EGF can induce BMSCs differentiate into neural cells. In the serum-free condition, BMSCs tend to differentiate into neurons, while under high serum concentration tend to glial cells.