Wu Ying-chen, Cui Lei, Li Gang, Yin Shuo, Gao Yong-juan, Cao Yi-lin
Department of Plastic Surgery, Shanghai Ninth People's Hospital, Shanghai Tissue Engineering Research & Development Center, Shanghai 200211, China.
Zhonghua Zheng Xing Wai Ke Za Zhi. 2007 Jul;23(4):335-9.
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).
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 alpha-actin, SM calponin, SMMHC and SM 22alpha were detected by immunofluorescence and observed with fluorescence microscope. mRNA expression of SMalpha-actin, SM calponin, SMMHC as well as SM 22alpha was analyzed by RT-PCR. The method of Western-Blot was applied to determine protein expression of SM 22alpha. Cells with induction were observed for the expression of SM alpha-actin,SM calponin,SMMHC by FACs analysis.
With the induction of PDGF-BB, the morphology of cells changed to a spindle fibroblastic appearance. By fluorescence microscope observation, expression of SM alpha-actin, SM calponin and SMMHC was found intracellularly in PDGF-BB treated hBMSCs at 14 days. Western-Blot detection confirmed SM 22alpha expression by 14 days induction. RT-PCR of characteristic vascular smooth muscle cells related genes, such as SM alpha-actin, SM calponin, SMMHC and SM 22alpha revealed differentiation of vascular smooth muscle cells phenotype in monolayer culture upon stimulation with PDGF-BB for 14 days. The positive expression of SM alpha-actin, SM calponin and SMMHC in induced cells was significantly higher than that in non-induced cells (P < 0.05, n=3).
These results suggested hBMSCs could be differentiated into vascular smooth muscle cell phenotype with PDGF-BB induction in vitro.
探讨人骨髓间充质干细胞(hBMSCs)在血小板衍生生长因子BB(PDGF-BB)诱导下体外分化为血管平滑肌细胞的可行性。
从成年健康供体的髂嵴穿刺获取骨髓间充质干细胞,在低糖杜氏改良伊格尔培养基(DMEM-LG)中扩增。第1代细胞转移至含PDGF-BB(20 ng/ml)的内皮细胞生长培养基-2(EGM-2)中培养14天。通过免疫荧光检测平滑肌α肌动蛋白(SMα-actin)、平滑肌钙结合蛋白(SM calponin)、平滑肌肌球蛋白重链(SMMHC)和SM-22α的表达,并在荧光显微镜下观察。采用逆转录-聚合酶链反应(RT-PCR)分析SMα-actin、SM calponin、SMMHC以及SM-22α的mRNA表达。应用蛋白质免疫印迹法(Western-Blot)检测SM-22α的蛋白表达。通过流式细胞术(FACs)分析诱导后的细胞中SMα-actin、SM calponin、SMMHC的表达情况。
在PDGF-BB诱导下,细胞形态变为纺锤形的成纤维细胞样外观。通过荧光显微镜观察,发现经PDGF-BB处理14天的hBMSCs细胞内有SMα-actin、SM calponin和SMMHC的表达。蛋白质免疫印迹检测证实诱导14天后有SM-22α表达。对特征性血管平滑肌细胞相关基因如SMα-actin、SM calponin、SMMHC和SM-22α进行RT-PCR检测,结果显示在PDGF-BB刺激14天的单层培养中,出现了血管平滑肌细胞表型的分化。诱导细胞中SMα-actin、SM calponin和SMMHC的阳性表达显著高于未诱导细胞(P<0.05,n = 3)。
这些结果表明,hBMSCs在体外经PDGF-BB诱导可分化为血管平滑肌细胞表型。