骨髓基质细胞向血管内皮细胞分化的体外培养研究

In vitro culture of bone marrow stromal cells and their differentiation into vascular endothelial cells

目的研究骨髓基质细胞(marrowstromalcells,MSCs)向血管内皮细胞分化的可能性,以及内皮细胞特异性标志的表达。方法实验选用7周龄清洁级雄性SD大鼠4只,抽取SD大鼠骨髓,进行体外培养和连续传代,获得较纯的MSCs。采用第4代的MSCs,以内皮细胞无血清条件培养液,表皮细胞生长因子(epidermalgrowthfactor,EGF)、碱性成纤维细胞生长因子(basicfibroblastgrowthfactor,bFGF)进行体外诱导,相差显微镜下观察各组细胞形态变化,以免疫组化方法鉴定内皮细胞特征样蛋白VIII因子的表达阳性率,并通过透射电镜观察内皮细胞特异性W-P小体。结果诱导前MSCs多呈扁平、多突起形态,诱导后形态均发生变化,10d细胞呈扁圆形,20d后细胞之间渐呈集落状、铺路石样排列。免疫组化检测VIII因子阳性比例接近70%。透射电镜显示诱导后细胞内出现内皮细胞特异性W-P小体。结论成体中的MSCs在一定诱导条件下可以表现内皮细胞样特征,是临床治疗缺血性疾病理想的移植用种子细胞。

AIM:To explore the possibility of differentiation of bone marrow stromal cell(MSC) into vascular endothelial cells,and to study the specific signaling expression of endothelial cells.METHODS:Four 7 week clean male SD rats were selected.Their bone marrow cells were extracted,cultured in vitro and continuous transfection to get purer MSC.MSC in the fourth generation was used.After induction in vitro with the endothelial cell non serum conditioned medium,epidermal growth factor(EGF) and basic fibroblast growth factor(bFGF),morphologic change of cells was observed under phase contrast microscope.Positive rate of the expression of protein VIII factor in endothelial cells was evaluated by immunohistochemistry,and W P corpuscle was observed by transmission electron microscope.RESULTS:Before induction,MSCs presented as thin and flat,with processus;While 10 days after induction,the cells were oblate,20 days later,they arrayed as gathering dropping and paving shape.Rate of VIII positive cells was close to 70%by immunohistochemistry test.W P corpuscle was observed by transmission electron microscope after induction in the endothelial cell.CONCLUSION:The MSCs presented features of endothelial cells in a certain induction condition in the adults.They are ideal donor cells for the ischemic diseases treatment.