用于造血干细胞移植的人骨髓间充质干细胞体外扩增研究

In Vitro Expansion of the Adult Human Bone Marrow Mesenchymal Stem Cells for Clinic Application in HSCT

本研究旨在检查4种不同培养液体外培养人骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM-MSCs)的效果,建立一种基于临床移植需要的分离、培养人BM-MSC的方法,为BM-MSC联合造血干细胞移植的临床应用提供实验基础。采用Ficoll Paque细胞分离液从16例新鲜成人骨髓穿刺液中分离BM-MSC,分别用含10%脐带血血清、胎牛血清、成人血清的DMEM/F12培养液和MesenCult培养液培养,用流式细胞术检测细胞表面抗原,以成骨诱导体系及脂肪诱导体系分别诱导各组BM-MSC定向分化,通过细胞形态观察、免疫表型及免疫化学染色进行鉴定。结果表明:4种培养液都能从成人骨髓液中分离培养出BM-MSC,脐带血血清组和MesenCult组培养的BM-MSC在增殖数量和速度上相似,但均优于FCS组和AB型血清组。脐带血血清组和MesenCult组培养的BM-MSC表达CD29、CD73、CD105的阳性率比FCS组和AB型血清组高(p(0.05),而CD31阳性率低于FCS组和AB型血清组(p(0.05),脐带血血清组和MesenCult组培养的BM-MSC诱导为成骨细胞和脂肪细胞阳性率高于FCS组和AB型血清组(p(0.05)。结论:4种不同培养液能够从成人骨髓液中分离出BM-MSC,脐带血血清组和Mes-enCult组培养的BM-MSC的纯度和体外诱导分化能力比FCS组和AB型血清组高,含有脐带血血清的培养体系具有临床应用价值。

This study was aimed to investigate the efficiency of 4 different culture media for in vitro culture and expanding adult human bone marrow mesenchymal stem cells （ahBM-MSCs） so as to establish a protocol of culturing and expanding hBM-MSCs and provide exprimental basis for hematopoietic blood stem cell transplantation combined with BM-MSCs. BM-MSCs were obtained from 16 fresh adult human bone marrow aspirate by gradient centrifugation with Ficoll Paque, then cultured in 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 were differentiated into osteoblasts and adipocytes under culture in the conditioned medium special for osteogenesis, and adipogenesis and the differentiated MSCs were identified by morphological observation, immunophenotype and immunohistochemical staining. The results showed that BM-MSCs could be isolated from adult human bone marrow and cultured by all culture media. The effect of umbilical cord blood serum on BM-MSC proliferation and their purity were similar to that of MesenCult culture medium, but better than that of FCS and human blood serum. The positive rate of CD29, CD73, CD105 on BM-MSCs cultured in umbilical cord serum and MesenCult medium was higher than that in FCS and adult human serum （p 〈 0.05 ）, and the positive rate of CD31 was lower than that in FCS and adult human serum （p 〈 0.05 ）. The positive rate of BM-MSCs differentiated into osteoblasts and adipocytes under culture in the conditioned medium for osteogenesis and adipogenesis with umbilical cord blood serum and MesenCult culture medium was also higher than that in FCS and adult human serum （p 〈 0.05 ）. It is concluded that BM-MSCs can be obtained by all the four methods. DMEM/F12 with 10% umbilical cord blood serum and MesenCult culture medium are better than the others for the purification and differentiation potency of BM-MSCs in vitro. The medium with umbilical cord serum is valuable for clinical application in HSCT.