无血清条件下骨髓间充质干细胞增殖及分泌VEGF的实验研究

Experimental study of proliferation and vascular endothelial growth factor expression of bone marrow mesenchymal stem cells under serum-free medium

目的探讨大鼠骨髓间充质干细胞(BMSCs)在不同无血清培养时间下增殖及分泌血管内皮生长因子(VEGF)的规律。方法采用贴壁培养法纯化BMSCs,流式细胞仪鉴定第3代(P3)BMSCs的细胞表型;比较BMSCs在不同无血清培养时间下的细胞凋亡率;将BMSCs分为无血清培养12、24、36、48及72h组,用ELISA方法检测VEGF的分泌。结果原代BMSCs培养24h后贴壁生长良好;经流式细胞仪检测,BMSCs表面CD34、CD45阴性,而CD29、CD44、CD90阳性,符合BMSCs特征;无血清培养12、24、36、48、72h后细胞凋亡率组间比较,差异无统计学意义(P>0.05),培养96h后凋亡率与其余各组比较差异有统计学意义(P<0.05);随着无血清培养时间的延长,BMSCs分泌VEGF逐渐增多(P<0.05),无血清培养72h组分泌VEGF水平最高。结论大鼠BMSCs在无血清培养条件下,72h内细胞凋亡无明显增多,且随着无血清培养时间的延长,分泌VEGF逐渐增多。

Objective To explore the law that BMSCs proliferation and secretion of VEGF under serum-free medium by different cultured time.Methods Using the adherent method to purify BMSCs,cell phenotypes of third-generation(P3) BMSCs were detected by flow cytometry.Compare the cell apotosis rate of BMSCs under serum-free medium by different cultured time;Acording to the serum-free culture time.BMSCs were divided into 12,24,36,48 and 72 h group,then using ELISA method to detection the secretion of VEGF.Results After 24 h culturation,primary BMSCs grew well.Cell phenotypes of BMSCs by flow cytometry,showing CD34,CD45 negative,while CD29,CD44,CD90 positive,in line with BMSC features.The cells apotosis rate of BMSCs cultured under serum-free medium by 12,24,36,48 and 72 h were no significant difference(P<0.05),but had statistics difference by 96 h(P>0.05).BMSCs had secreted the highest level of VEGF under serum-free medium of 72 h group,and 12 h group<24 h group<36 h group<48 h group<72 h group(P<0.05).Conclusion Cells apotosis rate no significant increase within 72h in serum-free culture conditions,and according to the serum-free culture time increased,secretion of VEGF increased too.So this experimental results laiding the foundation for the next experiment.