| 摘要: |
| 目的：对体外分离培养脐血间充质干细胞（UCB-MSCs）的传统方法进行改进，以提高培养成功率，同时观察其生物学特性。方珐：无菌条件下取28份足月产新生儿脐血，以密度梯度离心法分离其中的单个核细胞，以含10％FBS的α-MEM培养基进行体外培养。原代培养5～7d后半量换液，后每隔3～4d全量换液1次。细胞贴壁之后按改良方法进行培养：方法一，当皿底圆形巨核细胞融合、梭形成纤维样细胞脱落时将细胞悬液移入新的皿中培养；方法二，待皿底圆形巨核细胞渐渐占取优势时，将培养基换为含15％FBS的α-MEM，当圆形巨核细胞大部脱落后换回含10％FBS的α-MEM培养基。显微镜下观察UCB-MSCs的形态，流式细胞仪测定细胞免疫表型，体外成骨及成脂诱导并进行细胞化学染色。结果：28份脐血中20份培养出贴壁细胞，其中13份培养出能融合且可稳定传代的成纤维样细胞，成功率为46．4％，可传至22代而无形态上的变化，而且强烈表达CD105、CD29等MSCs表面标志，而CD34、CD45和CDl06等表达呈阴性。在特定条件下，UCB-MSCs可分化为成骨细胞和脂肪细胞。结论：脐血中存在MSCs，培养方法经改良后可提高UCB-MSCs的培养成功率。UCB-MSCs具有与其他来源的MSCs类似的表型及分化潜能，且易于体外扩增、传代稳定，可望在骨组织工程学研究中有广阔的应用前景。 |
| 关键词: 脐血 间充质干细胞 细胞 培养 免疫表型分型 |
| DOI：10.3724/SP.J.1008.2007.00027 |
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| 基金项目: |
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| Improvement of in vitro culture method for umbilical cord blood mesenchymal stem cells and study of biological characteristics of cultured cells |
| WANG Qi GOU San-huai , TANG Ting-ting , LIU Yan, OUYANG Yue-ping, XI Yan-hai , H AN Qing-lin |
| (1. Department of Orthopaedics, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China;2. Department of Orthopaedics, the 9^th People＇ s Hospital of Shanghai, Shanghai Jiaotong University, Shanghai 200011) |
| Abstract: |
| Objeetive ： To improve the traditional in vitro culture method for umbilical cord blood mesenchymal stem cells （UCB-MSCs） in an effort to increase the successful culture rate and to study the biological characteristics of the cultured cells. Methods. Full-term UCB samples were obtained with the mothers＇ consent and processed within 12 h of collection （n= 28）. Mononuclear cells （MNCs） were isolated from UCB by centrifugation at 2 500 r/min for 20 rain and were suspended in presence of a-minimum essential medium（α-MEM） containing 10% fetal bovine serum （FBS） and 100 U/ml penicillin. The medium was half changed after 5-7 days＇ primary culture and then was totally changed at a .3-4 days＇ interval. After the cells adhered to the culture wall, the following procedures were carried out according to our improved methods. In group Ⅰ , the suspension containing detached mesenchymal-like cells were removed onto a new culture dish when osteoclast-like cells reached over 80% confluence. In group Ⅱ , 15% calf serum instead of 10% FBS was used in the culture medium when osteoclast-like cells were growing confluence, and α-MEM medium containing 10% PBS was used again until most of the osteoclast-like cells were detached. The morphology of UCB-MSCs was observed under microscope and the phenotypes of them were examined by flow cytometry. The 5＇h passage cells were cultured with osteogenic medium and adipogenic medium separately and cytochemical staining was carried out subsequently. Results： Adhered cells were cultured from 20 of the 28 samples. UCB-MSCs were cultured from 13 of the 20 samples, which could grow to confluence and could be stably passaged to P22. The cultured UCB-MSCs were all positive for MSC-related antigens, such as CD29 and CD105, but negative for CD34, CD45, and CD106. We also found that the cultured UCB-MSCs could differentiate into osteoblasts and fat cells under specified conditions. Conclusion： MSCs do exist in UCB, and improvement of culture method can increase the yielding of UCB-MSCs. The cultured cells, with the similar phenotype and differentiation potential as those derived from other resources, can be amplified easily and passaged stably in vitro, making them promising seed cells for future bone tissue engineering |
| Key words: umbilical cord blood mesenchymal stem cells cells, cultured immunophenotyping |
