体外诱导骨髓间充质干细胞向心肌样细胞分化

In vitro induction of differentiation of bone marrow derived mesenchymal stem cells to cardiomyocytelike cells

目的 探讨生长因子在体外能否诱导大鼠骨髓来源的间充质干细胞（BM-MSC）向心肌样细胞分化,是否增强其旁分泌作用.方法 取4周龄雄性SD大鼠骨髓,贴壁法培养BM-MSC至第3代时,应用流式细胞仪检测其表面标志物CD29、CD44、CD105、CD34、CD45、CD31.将细胞随机分为间充质干细胞组（MSC组）和生长因子共培养组（GF-MSC组）.MSC组继续培养传代;GF-MSC组与生长因子[其中包括培养基（IMDM）、2％胎牛血清（FBS）、20 nmol/L地塞米松、100μmo1/L维生素C（VitC）、50 μg/L成纤维细胞生长因子2（FGF-2）、10 μg/L成骨蛋白2（BMP-2）、2μg/L胰岛素样生长因子1（IGF-1）、青霉素和100 mg/L链霉素]共培养.1周后细胞免疫荧光染色检测两组细胞肌钙蛋白Ⅰ（TnⅠ）和结蛋白（Des）表达;反转录-聚合酶链反应（RT-PCR）检测两组细胞Tn Ⅰ和Des mRNA的表达;ELISA法检测两组细胞裂解液以及细胞培养上清中的肝细胞生长因子（HGF）和血管内皮生长因子（VEGF）的蛋白水平.结果 体外培养的BM-MSC表达CD29、CD44、CD105,而不表达CD31、CD34、CD45.免疫荧光染色显示GF-MSC阳性表达TnⅠ和Des.RT-PCR结果显示TnⅠ和Des mRNA在GF-MSC有阳性表达,而MSC为阴性表达.GF-MSC组HGF、VEGF在细胞裂解液和细胞培养上清中的蛋白水平（ng/L）高于MSC组（HGF裂解液32.05±0.41比12.10±0.21,培养上清574.21±4.19比169.16±2.41;VEGF裂解液45.66±2.22比30.37±0.54,培养上清487.78±36.29比44.25±1.31;均P＜0.05）.结论 体外与生长因子共培养,可诱导BM-MSC向心肌样细胞分化,并增强其旁分泌作用.

Objective To investigate whether growth factors in vitro can induce rat bone marrow derived mesenchymal stem cells （BM-MSCs） differentiation to cardiomyocyte like cells,and enhance paracrine effect of BM-MSC.Methods The MSCs were isolated from the marrow of femur of 4-weeks old male SD rats and cultured.Cell surface markers（CD29,CD44,CD105,CD34,CD45,CD31） of BM-MSCs of the third passage were analyzed by flow cytometry.Cells were randomly divided into MSCs group and GF-MSCs group.MSCs group was cultured normally while GF-MSCs group was co-cultured with growth factors,including medium （IMDM）,2％ fetal bovine serum （FBS）,20 nmol/L dexamethasone,100 μmol/L vitamin C （VitC）,50 μg/L fibroblast growth factor-2 （FGF-2）,10 μg/L osteogenic protein 2 （BMP-2）,2 μg/Linsulin-like growth factor 1 （IGF-1）,penicillin,and 100 mg/L streptomycin.After 1 week culture,protein expressions of troponin Ⅰ （Tn Ⅰ） and desmin （Des） were detected in both groups by immunofluorescence staining.Tn Ⅰ and Des mRNA expressions were detected by reverse transcription-polymerase chain reaction （RT-PCR）.Release of hepatocyte growth factor （HGF） and vascular endothelial growth factor （VEGF） in cell lysates and cell culture supernatants from both groups was measured by ELISA assay.Results Cultured BM-MSCs expressed CD105,CD44,CD29,but not CD31,CD34 and CD45.GF-MSCs group showed positive expression of cardiac Tn Ⅰ and Des by immunofluorescence assay and RT-PCR,while MSCs group showed negative.Protein concentration levels （ng/L） of HGF and VEGF in cell lysates and cell culture supernatant were significantly higher in GF-MSCs group compared to MSCs group （HGF lysates：32.05±0.41 vs 12.10±0.21,cell culture supernatant 574.21±4.19 vs 169.16±2.41 ; VEGF lysates：45.66±2.22 vs 30.37± 0.54,cell culture supernatant 487.78±36.29 vs 44.25±1.31,all P＜0.05）.Conclusion Co-culture of BMMSCs with growth-factors in vitro can induce to cardiomyocyte like cell differentiation and enhances the paracrine effect of MSCs.