非接触共培养法诱导人骨髓间充质干细胞向内皮细胞的分化

Inducing human bone marrow mesenchymal stem cells to differentiate into endothelial cells with indirect co-culture

背景:最近研究表明将乳鼠心肌细胞和骨髓间充质干细胞非接触共培养后,可成功将骨髓间充质干细胞诱导分化为心肌细胞。目的:尝试通过非接触共培养方式,利用人脐静脉内皮细胞诱导人骨髓间充质干细胞向内皮细胞分化。设计、时间及地点:细胞组织工程学体外实验,于2007-01/07在广东省人民医院医学研究中心完成。材料:骨髓标本来源于11例无血液系统疾病的先天性心脏病患儿,征得家属同意后在心脏矫治手术中从胸骨柄穿刺获取少量骨髓用于实验。足月顺产健康新生儿脐带由中山大学附属一院产科协助取得。新鲜牛颈静脉由广东大沥菜牛屠宰厂提供。方法:取骨髓标本,以密度梯度离心法分离纯化人骨髓间充质干细胞,并在体外扩增培养;以酶消化法自新生儿脐带获取人脐静脉内皮细胞。采用具有半透膜的细胞培养池结合6孔板的方式进行非接触共培养诱导,将人脐静脉内皮细胞铺于培养池内,第3代骨髓间充质干细胞以1×105/孔铺在培养池外的6孔板内,两种细胞的初始比例为1:5,加入含体积分数为0.1胎牛血清的低糖DMEM培养液,培养14d。以骨髓间充质干细胞和骨髓间充质干细胞共培养作为对照组。诱导后的内皮样细胞扩增培养,种植在脱细胞牛颈静脉血管支架上。主要观察指标:诱导后细胞形态变化,免疫细胞化学染色检测诱导后内皮样细胞表面相关抗原,扫描电镜观察内皮样细胞在血管支架上的生长黏附情况。结果:诱导后的内皮样细胞形态均一,呈铺路石状排列,扩增迅速,表达内皮细胞特有的表面标志CD31和vWF,阳性率均>99%,可以在脱细胞牛颈静脉血管支架表面形成连续的单细胞层。结论:非接触共培养方式下,人脐静脉内皮细胞可成功诱导人骨髓间充质干细胞向内皮细胞分化,并能够在脱细胞牛静脉血管支架上黏附生长。

BACKGROUND： Recent studies indicate that after indirect co-culture of neonate rat myocardial cells and bone marrow mesenchymal stem cells, bone marrow mesenchyrnal stem cells can differentiate into myocardial cells successfully. OBJECTIVE： To induce human bone marrow mesenchymal stem cells （hBMSCs） to differentiate into endothelial cells using human umbilical vein endothelial cells by indirect co-culture. DESIGN, TIME AND SETTING： In vitro study of cell engineering was done at the Medical Research Center of Guangdong Provincial People＇s Hospital between January and July 2007. MATERIALS： Small quantities of bone marrow were obtained from 11 children with congenital heart disease but without hematologic diseases through manubrium of sternum puncture in the congenital heart defect corrective surgery after the permission of family member. Umbilical cord of full-term normal delivery healthy newborn was provided by the Department of Obstetrics, the First Affiliated Hospital of Sun Yat-sen University. Fresh cattle jugular vein was provided by Guangdong Dali Meat Cattle Butchery METHODS： The hBMSCs were isolated and purified using density gradient centrifugation method and were cultured in vitro. Human umbilical vein endothelial cells were obtained from newborn umbilical cord by enzyme digestion. Cell culture insert with semipermeable membrane combined with 6-well plate was used to do indirect co-culture induction. Human umbilical vein endothelial cells were expanded in the cell culture insert, passage 3 bone marrow mesenchymal stem cells were expanded in the 6-well plate outside the culture insert at the density of 1 × 10^5 cells/well, the initial ratio of the two kinds of cells was 1：5, then low-glucose DMEM culture solution containing 10% fetal bovine serum was added, cells were cultured for 14 days. Co-culture of bone marrow mesenchymal stem cells and bone marrow mesenchymal stem cells was used as control. Introduced endothelioid cells were cultured and then seeded on the cell-free cattle jugular vein intravascular stent. MAIN OUTCOME MEASURES： Morphology changes of induced cells; introduced endothelioid cell surface antigen detected through immunocytochemical staining; the growth and adhesion condition of endothelioid cells on the intravascular stent observed under scanning electronic microsope. RESULTS： The morphologies of introduced endothelial cells were uniform, introduced endothelial cells presented a cobblestone-like appearance, they amplified fast and expressed endothelial cell-specific surface marker CD31 and vWF and the positive rate was over 99%. They also could form a continuous unicellular layer on the cell-free cattle jugular vein intravascular stent. CONCLUSION： Human umbilical vein endothelial cells can induce hBMSCs to differentiate into endothelial cells successfully and to adhere and grew on the cell-flee cattle jugular vein intravascular stent through indirect co-culture method.