体外诱导人骨髓间充质干细胞向血管平滑肌细胞分化的实验研究  

PDGF-BB initiates vascular smooth muscle-like phenotype differentiation of human bone marrow mesenchymai stem cells in vitro

作　　者：吴莹琛[1] 崔磊[1] 李纲[2] 尹烁[2] 高永娟[2] 曹谊林[1] 

机构地区：[1]上海第二医科大学附属第九人民医院整形外科,200011 [2]上海组织工程研究与开发中心

出　　处：《中华整形外科杂志》2007年第4期335-339,共5页Chinese Journal of Plastic Surgery

基　　金：霍英东青年教师基金（2004-2007）;上海市教委“曙光计划”课题（2005-2008）;上海市2005年度重大基础研究项目（05DJ14005）

摘　　要：目的应用血小板衍生生长因子BB（platelet-derived growthfactor BB,PDGF-BB）,体外诱导人骨髓间充质干细胞（human bone marrowmesenchymal stemcells,hBMSCs）向血管平滑肌细胞表型分化,探讨该方法的可行性及诱导细胞作为组织工程血管平滑肌种子细胞的可行性。方法抽取健康成人志愿者骨髓,经密度梯度离心分离得单个核细胞,PDGF-BB（20ng/ml）诱导hBMSCs向血管平滑肌样细胞（vascular smooth muscle cells,VSMCs）分化,观察细胞形态变化。免疫荧光检测细胞内血管平滑肌肌动蛋白α（vascular smooth muscleα-actin,SMα-actin）,血管平滑肌钙结合蛋白（vascular smoothmuscle calponin,SMcalponin）,血管平滑肌肌球蛋白重链（vascular smooth muscle myosin heavy chain,SMMHC）和细胞血管平滑肌钙结合相关蛋白（smooth muscle22α,SM22α）表达情况;反转录聚合酶链式反应（RT-PCR）检测诱导后血管平滑肌细胞SMα-actin,SMcalponin,SMMHC和SM22α的mRNA表达。Western印记检测SM22α的表达。流式细胞分析技术（fluorescence activated cell sorter,FACS）分析诱导后细胞内SMα-actin,SMcalponin,SMMHC的表达。结果PDGF-BB20ng/ml诱导后可见单层培养的细胞形态呈“成纤维细胞样”。免疫荧光检测示SMα-actin,SMcalponin,SMMHC,SM22α表达阳性;RT-PCR检测SMα-actin,SMcalponin,SMMHC,SM22α的mRNA阳性表达。Western印迹检测SM22α的表达为阳性。FACS分析表明诱导后,SMα-actin,SMcalponin,SMMHC表达均增高,与未诱导组比较差异有统计学意义（P〈0.05,n=3）。结论人骨髓间充质干细胞在PDGF-BB的诱导下可向血管平滑肌细胞表型分化,有望成为血管组织工程血管平滑肌种子细胞的来源。Objective To investigate the feasibility of human bone marrow mesenchymal stem cells （hBMSCs） in vitro differentiation into vascular smooth muscle cells with induction of platelet-derived growth Factor BB （PDGF-BB）. Methods Bone marrow mesenchymal stem cells of adult healthy donors were separated from iliac crest aspiration and expanded in DMEM-LG medium. Cells at passage 1 were transferred to EGM-2 medium containing PDGF-BB （20 ng/ml ） and cultured for 14 days. The expression of SM α-actin,SM calponin, SMMHC and SM 22a were detected by immunofluorescence and observed with flurescence microscope, mRNA expression of SMα-actin,SM calponin, SMMHC as well as SM 22a was analyzed by RT-PCR. The method of Western-Blot was applied to determine protein expression of SM 22α. Cells with induction were observed for the expression of SM α-actin,SM calponin,SMMHC by FACs analysis. Results With the induction of PDGF-BB, the morphology of cells changed to a spindle fibroblastic appearance. By fluorescence microscope observation, expression of SM α-actin, SM calponin and SMMHC was found intracellularly in PDGF-BB treated hBMSCs at 14 days. Western-Blot detection confirmed SM-22a expression by 14 days induction. RT-PCR of characteristic vascular smoooth muscle cells related genes, such as SM α-aetin, SM calponin, SMMHC and SM-22α revealed differentiation of vascular smooth muscle cells phenotype in monolayerd culture upon stimulation with PDGF-BB for 14 days. The positive expression of SM α-actin, SM calponin and SMMHC in induced cells was significantly higher than that in non-induced cells （P 〈 0.05, n = 3）. Conclusion These results suggested hBMSCs could be differentiated into vascular smooth muscle cell phenotype with PDGF-BB induction in vitro.

关 键 词：人骨髓间充质干细胞 血管平滑肌细胞 分化 血小板衍生生长因子BB 

分 类 号：R686[医药卫生—骨科学]