脐带血清体外培养临床应用人骨髓间充质干细胞的方法研究

Study on in vitro expansion the adult Human bone marrow mesenchymal stem cells from umbilical cord blood serum

目的:观察脐带血清等4种不同培养体系体外培养人骨髓间充质干细胞(BM-MSCs)的效果,建立一种适合临床移植需要的人BM-MSCs培养方法。方法:从20例新鲜成人骨髓穿刺液中分离BM-MSCs,分别用含10%脐带血清、胎牛血清(FCS)、成人血清的DMEM/F12培养基和MesenCult培养基培养,流式细胞仪检测细胞表面抗原,成骨诱导体系及脂肪诱导体系分别诱导各组BM-MSCs定向分化。结果:采用4种培养体系都能从骨髓液中分离培养出BM-MSCs,脐带血清和MesenCult组培养的BM-MSCs在增殖数量和速度上相似,而优于FCS和AB型血清组。脐带血清和MesenCult组培养的BM-MSCs表达CD29、CD73、CD105的阳性率比FCS和AB型血清组高(均P<0.05),而CD31阳性率低于FCS和AB型血清组(P<0.05),脐带血清和MesenCult组培养的BM-MSCs诱导为成骨细胞和脂肪细胞阳性率高于FCS和AB型血清组(P<0.05)。结论:脐带血清与MesenCult组培养的BM-MSCs的纯度和体外诱导分化能力相似,优于FCS和AB型血清组,脐带血清适合临床应用。

Objective： Observe four culture methods of BM-MSCs. To establish a protocol of culturing adult human bone marrow mesenchymal stem cells （BM-MSCs）. Method..BM-MSCs were obtained from 20 fresh adult human bone marrow and respectively cultured by DMEM/F12 with 10% umbilical cord blood serum, 10 % fetal calf serum （FCS）, human blood serum, and MesenCult culture medium. The surface antigens of BM-MSCs were detected by flow cytometry. BM-MSCs would differentiate into osteoblasts and adipocytes in conditioned medium. Result：BM-MSCs from adult human bone marrow could be isolated and cultured by four culture medium. The effect of umbilical cord blood serum on BM-MSCs proliferation and purity was similar to MesenCult medium, better than FCS and human blood serum. The positive rates of CD29, CD73, CD105 on BM-MSCs cultured in umbilical cord serum and MesenCult medium were higher than those in FCS and adult human serum （ P 〈0.05） whereas the positive rate Of CD31 was lower than it in FCS and adult human serum （ P 〈0.05）. The positive rate of osteoblasts and adipocytes differentiated from BM-MSCs in cord blood serum and MesenCult medium was also higher than those in FCS and adult human serum （ P 〈0. 05）. Conclusion： Umbilical cord blood serum is similar with MesenCult medium , but superior to the FCS and adult human serum both in the purification and differentiation potency of BM-MSCs. The umbilical cord serum is suitable for clinical use.