改良法体外分离培养成年大鼠骨髓间充质干细胞实验研究

目的寻找一个稳定、快速、获取大量高纯度的成年骨髓间充质干细胞(BMSCs)的实验方法,为组织工程种子细胞来源提供实验依据。方法使用全骨髓贴壁法分离成年大鼠BMSCs,设立含10%胎牛血清(FBS)的DMEM/F12培养组为对照组,含10%FBS、10ng/mL表皮生长因子(EGF)的DMEM/F12培养组为实验组,倒置显微镜下观察比较两组原代、传代后BMSCs的形态及生长情况,使用流式细胞仪检测实验组P6、P10细胞表面标记。结果实验组原代细胞达融合的时间为5～6d,对照组为7～9d。传代后实验组细胞贴壁伸展的时间为2h,而对照组为6h。对照组p1细胞达融合时间为5～6d,实验组P1细胞达融合时间为4d,流式细胞仪检测实验组P6、P10BMSCs,发现P6细胞92%细胞表达CD29,3.5%细胞表达CD44,有14%的细胞表达CD45;而P10BMSCs抗原表达具有较高的均一性,CD29/CD44强阳性表达,仅有2%的细胞表达CD45。结论 10%FBS、10ng/mL表皮生长因子(EGF)的DMEM/F12全骨髓贴壁法培养BMSCs能够稳定、简便、快速获取大量高纯度成年大鼠BMSCs,满足组织工程对种子细胞的要求。

The experimental study on the improved method of adult rat BMSCs

Objective To obtain highly purified and large amount of adult rat Bone Mesenchymal stem cells(BMSCs) by improved culture method for nerve tissue engineering.Methods Based on the previous experiment,the improved culture medium combined with whole bone marrow culture was used,establishing two groups.One is experimental group used 10% FBS、10ng /ml EGF of DMEM /F12,and the other is control group with 10% FBS DMEM /F12 in this study.The morphologic features and growth condition primary and passaged cells in two groups were observed under inverted microscope.The P6 and P10 BMSCs was tested by flow cytometry.Results The confluence of primary cells was happened within 5-6 days in experimental group,but within 7-9 days in control group.The extention of passaged cells was 2 h and the confluence of P1 cells was happened in 4 days in experimental group,accordingly 6h and within 5-6 days in control group.The results of Flow Cytometry showed that the 92% P6 BMSCs in experimental group expressed CD29,that 3.5% cells expressed CD44 and 14% cells expressed CD45,while the P10 BMSCs in experimental group expressed CD29 and CD44 intensively but scarcely expressed CD45.Conclusion The whole bone marrow culture with 10% FBS,10 ng /mL EGF of DMEM /F12 can obtain large number of highly pure adult BMSCs in vitro rapidly,stably and effectively,which would meet the demand of tissue engineering.