转化生长因子-β1和血清对人骨髓基质干细胞增殖及向软骨细胞诱导分化的影响

Effects of TGF-β1 and serum on the proliferation and chondrogenic differentiation of human bone marrow stromal cells

目的探讨转化生长因子-β1(TGF-β1)和不同浓度胎牛血清对体外培养人骨髓基质干细胞(BMSCs)增殖及向软骨细胞诱导分化的作用,为软骨组织工程提供有用的种子细胞。方法分别采集3例志愿者骨髓各6mL,用Percoll细胞分离液分离,收集单个核细胞,在含10%胎牛血清的低糖DMEM培养液中培养扩增。将第3代细胞分为3组:A组为对照组,B组为含5%胎牛血清的诱导培养基组,C组为含10%胎牛血清的诱导培养基组。倒置显微镜下观察细胞生长状况,免疫组化检测Ⅱ型胶原分泌,原位杂交检测Ⅱ型胶原mRNA表达,3H标记的胸腺嘧啶脱氧核苷(3H-TdR)掺入实验检测细胞的增殖情况。结果经密度梯度离心后,活细胞比例在96%以上。贴壁后的细胞形态均一,由梭形向多角形转变。B组细胞在诱导培养7d后Ⅱ型胶原免疫组化均为阳性,原位杂交可检测到Ⅱ型胶原mRNA的表达,而A、C两组分别为阴性和弱阳性。三组细胞3H-TdR摄入量的大小顺序为C>B>A。结论密度梯度离心法是一种高效、可大量获取人BMSCs的方法,细胞在体外生长稳定;低浓度的胎牛血清和10ng/mL的TGF-β1联合作用既可促进人BMSCs的增殖,又可诱导其向软骨细胞方向分化,而高浓度胎牛血清仅对细胞的增殖有促进作用。

Objective To observe the effects of TGF-β1 and serum on the proliferation and chondrogenic differentiation of human bone marrow stromal cells （BMSCs） cultured in vitro. Methods A volume of 6 ml bone marrow was collected from 3 volunteer donors respectively. BMSCs were isolated from the Percoll fraction of mononuclear cells and cultured in the Dulbecco＇s Modified Eagle＇s Medium（DMEM） with low glucose containing 10% fetal bovine serum. BMSCs from the third passage were grown in complete medium （Group A）, inductive medium with 5% fetal bovine serum （Group B）, and inductive medium with 10% fetal bovine serum （Group C） respectively. The growth of the cultured BMSCs was observed, lmmunohistochemistry and in situ hybridization were applied to detect the expression of collagen type Ⅱ. The proliferation of the cells was examined by ^3H-TdR incorporation. Results 96% of the cells were alive after density gradient centrifugation. The adherent cells changed from a spindle-like appearance to a polygonal shape. After 7 days＇ induction by inductive medium with 5% fetal bovine serum, immunohistochemistry and in situ hybridization analysis proved strong type Ⅱ collagen expression. The sequences of the incorporation of 3H-TdR were C〉B〉A. Conclusions The density gradient centrifugation is an effective method to isolate BMSCs from bone marrow aspirate, for the BMSCs cultured in vitro grow stably. Combination of TGF-β1 and fetal bovine serum in low concentration （5%） can not only facilitate the proliferation of BMSCs, but also induce the chondrogenic differentiation.