不同种系鼠源骨髓间质干细胞的分离培养及成脂能力

摘要

背景:目前常用骨髓间质干细胞为鼠源细胞,然而就大鼠和小鼠来源骨髓间质干细胞在传代过程中的纯化程度,表面抗原的表达和分化能力的差异性仍少见报道.目的:分离、鉴定并纯化不同种系鼠源骨髓间质干细胞,比较其在成脂分化中的差异.方法:采用差速贴壁法分离SD大鼠及Balb/c小鼠骨髓间质干细胞,通过传代纯化,并观察骨髓间质干细胞在此过程中得纯化程度及增殖状况,流式细胞数鉴别两种骨髓间质干细胞表面抗原的表达,比较不同来源间质干细胞在成脂分化的差异.结果与结论:在SD大鼠骨髓间质干细胞分离和培养过程中,其增殖能力显著强于Balb/c小鼠骨髓间质干细胞(P < 0.05).其半数增长期为36 h,在第3代时即可见典型的漩涡状贴壁生长特性.经流式细胞检测,Balb/c小鼠及SD大鼠均为不表达造血干细胞标志CD34,再后续的各代检测中亦未见该细胞标志表达(< 5%),CD29随着不断的传代纯化其表达数量不断的增加,其中SD大鼠在第3代时其表面标志的纯度及达到99%,但Balb/c小鼠各代CD29随着纯化上升程度并不甚明显,与该种细胞镜下存在大量类内皮细胞表现相符合.在成脂方面SD大鼠骨髓间质干细胞依旧表现出了较强的多能特新,出现脂滴的时间明显早于Balb/c小鼠,随传代次数的增加,两种原代细胞均出现了自发的成脂分化的现象,且SD大鼠的出现程度大于Balb/c小鼠.说明SD大鼠来源骨髓间质干细胞较易获得纯度较高且增殖旺盛的细胞源,在成脂方面的能力强于Balb/c小鼠.%BACKGROUND: At present, the most commonly used bone marrow derived mesenchymal stem cells (BMSCs) are murine cells. However, the reports on the purification, expression of surface antigen and the differentiation ability of the BMSCs from rat and mice are rare.OBJECTIVE: To isolate, identify and purify different sources of BMSCs and to compare the difference on adipogenic differentiation.METHODS: The BMSCs were separated from SD rats and Balb/c mice by using differential adhesion, purified by passage, and the purification and proliferation of BMSCs through the process of status were observed. The expression of cell surface antigen of two kinds of BMSCs was identified by flow cytometry. The adipogenic differentiation ability between different sources of BMSCs was compared.RESULTS AND CONCLUSION: During isolation and culture of BMSCs, the proliferation of BMSCs from SD rats was significantly stronger than that from Balb/c mice (P < 0.05); the half of the growth period was 36 hours. The typical swirling posted wall growth characteristics could be seen in the third generation. Balb/c mice and SD rats could not express hematopoietic stem cells marker CD34 detected by flow cytometry, and there was no expression of the CD34 cell marker in the subsequent generations (< 5%), with the continuous passage purification, the number of CD29 expression was steady increased, the purity of the surface marker reached to 99% after SD rats BMSCs passaged to the third generation. But the increasing of CD29 was not very obvious in Balb/c mice BMSCs. This fact is consistent with endothelial like cells under the microscope. In adipogenic, SD rats BMSCs still showed a stronger multi-ability; the time for the lipid droplets in SD rats was significantly earlier than that in Balb/c mice. With the passage number increased, the spontaneous adipogenic differentiation phenomenon could be seen in two kinds of primary cells, and the adipogenic differentiation in SD rats BMSCs were stronger than that in the Balb/c mice BMSCs. It was easier to obtain high purity and proliferative source of cells from SD rats BMSCs. The adipogenic capacity of SD rats BMSCs was stronger than that of Balb/c mice BMSCs.