大鼠骨髓间充质干细胞向心肌样细胞诱导分化的体外条件研究

Rat Bone-marrow Mesenchymal Stem Cells Differentiate into Cardiomyocyte-like Cells under Diverse Conditions in vitro

目的研究体外条件下大鼠骨髓间充质干细胞(rat mesenchymal stem cells,rMSCs)向心肌样细胞分化的影响因素。方法采用免疫细胞化学方法检测rMSCs表面CD71、SH2、CD31、 CD34、CD45以及5-氮胞苷诱导后心肌细胞特异性蛋白α-sarcomeric actin、troponin T的表达;细胞计数法计算rMSCs倍增时间;观察不同传代数、不同时间及不同剂量5-氮胞苷、依地酸二钠(ethylenediami- netetraacetic acid tetrasodium salt,EDTA-2Na)及无血清培养液对rMSCs分化潜能的影响。结果rMSCs 表达CD71、SH2,而CD31、CD34和CD45呈阴性表达。第3代rMSCs经10μM 5-氮胞苷诱导后细胞呈现克隆、肌管等结,构,表达α-sarcomeric actin、troponin T等心肌细胞特异蛋白,细胞倍增时间为(100 ±8)h。第1、12代rMSCs诱导后细胞形态未发现明显改变、未出现上述结构,细胞倍增时间分别为 32±4 h、38±3 h。1、15、100μM 5-氮胞苷刺激12-72 h,2周后未发现克隆或肌管结构。5-氮胞苷刺激,以1 mM EDTA处理以及无血清培养液培养rMSCs形成的克隆、肌管结构不典型,α-sarcomeric ac- tin、troponin T表达较弱。结论rMSCs具有体外分化潜能,并且受细胞传代数、细胞倍增时间、诱导浓度和时间、细胞外钙离子浓度及培养条件等因素的影响。

Objective We investigated the effect of different factors on the differentiation of rat mesenchymal stem cells （rMSCs） in vitro. Methods rMSCs were obtained from femurs and tibias of male Sprague-Dawley （SD） rats and were isolated by Percoll density gradient centrifugation. We detected the expression for CD71, SH2, CD31, CD34, CD45 on a non-stimulation condition and cardiomyocytes specific proteins （ -sarcomeric actin and troponin T after 2 weeks induced by 5-azacytidine by immunocytechemistry. Passage 1, 3 and 12 of rMSCs were stimulated by 1, 3, 5, 10, 12, 15 and 100 μM 5-azacytidine for 12, 24, 48 and 72 hours to observe the effect of passage numbers, diverse dose and time of 5-azacytidine on the differentiation potential. Population doubling times of passage 1, 3 and 12 of rMSCs were estimated by cell counting to observe the effect of population number on the differentiation potential. Furthermore, the effects of EDTA-2Na（ ethylenediaminetetraacetic acid tetrasodium salt） and serum free DMEM on the differentiation potential were observed. Results CD71 and SH2 were positive expression in rMSCs, however CD31, CD34 and CD45 were negative. Two weeks after induction by 10 ixM 5-azacytidine for 24 hours, passage 3 rMSCs presented the specific clones and myotube appearances and were positive expressions for ot-sarcomeric actin and troponin T. The population doubling times of passage 1 and 12 rMSCs were less than that of passage 3, meanwhile we failed to observe the significant morphological transformations in passage 1 and 12 rMSCs, rM- SCs showed no dramatic clones or myotube after stimulated by 1, 15, 100 μM 5-azacytidine for 12 ~ 72 hours. In addition, in the present of 1 mM EDTA or serum-free culture medium, no dramatic clones or myo- tube structure transformations were observed and the expressions of ot-sarcomeric actin and troponin T were weak at 2 weeks after 5-azacytidine treatment. Conclusions These results indicated that many factors could affect the differentiation potential of rMSCs into cardiomyocytes-like cells in vitro, including cell passage numbers, population doubling times, the dose and time of 5-azacytidine, extracellular calcium and serum.