| 人脐带间充质干细胞分离扩增方法的优化 |
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| 引用本文： | 杨卿,杨扬,刘剑戎,潘国政,张英才,陈规划,张琪. 人脐带间充质干细胞分离扩增方法的优化[J]. 中山大学学报(医学科学版), 2011, 32(6): 818-823 |
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| 作者姓名： | 杨卿 杨扬 刘剑戎 潘国政 张英才 陈规划 张琪 |
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| 作者单位： | 中山大学附属第三医院肝移植中心∥广东省肝脏疾病研究重点实验室, 广东 广州 510630 |
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| 基金项目： | 中山大学青年老师培养项目，广东省科技计划项目，广州市科技计划支持项目 |
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| 摘 要： | 【目的】 探究一种高效稳定分离、扩增人脐带间充质干细胞（hUC-MSCs）的方法。【方法】取材脐带20条，获得Wharton’s Jelly组织，分别用酶联合消化法（12例）和酶序贯消化法（8例）来分离、提取hUC-MSCs，比较两种方法分离hUC-MSCs的成功率、原代培养时间及获得的细胞数量；利用低糖DMEM（LG-DMEM）完全培养基和LD-Mesen培养基（MesenPro RSTM与LG-DMEM的混合培养基）分别培养hUC-MSCs，比较两种培养体系的扩增效率。对所获取细胞的免疫表型以流式细胞术进行鉴定，并诱导成脂、成骨分化验证其多向分化潜能。【结果】 酶联合消化法和酶序贯消化法成功率分别为100%（12/12）和12.5%（1/8），二者相比有显著统计学差异（P=1.03×10-4），前者原代培养平均时间为（14.17±1.14）d，获得细胞（1.30±0.14）×106个。2×105个hUC-MSCs用LD-Mesen和LG-DMEM两种体系培养12d后，扩增所得的细胞数分别为(14.86±0.08)×106和(5.08±0.08)×106，二者有显著统计学差异（P=1.38×10-8)。hUC-MSCs高表达CD73、CD105、CD90、CD29、CD44，不表达CD31、CD34、CD45、HLA-DR；并可向成骨细胞及脂肪细胞诱导分化。【结论】 酶联合消化Wharton’s Jelly组织并以LD-Mesen体系进行扩增可高效获取hUC-MSCs，效率明显优于传统方法。
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| 关 键 词： | 间充质干细胞 脐带 细胞分离 |
| 收稿时间： | 2011-04-25; |
Optimized Isolation of Human Mesenchymal Stem Cells from Umbilical Cord |
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| YANG Qing,YANG Yang,LIU Jian-rong,PAN Guo-zheng,ZHANG Ying-cai,CHEN Gui-hui,ZHANG Qi. Optimized Isolation of Human Mesenchymal Stem Cells from Umbilical Cord[J]. Journal of Sun Yatsen University(Medical Sciences), 2011, 32(6): 818-823 |
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| Authors: | YANG Qing YANG Yang LIU Jian-rong PAN Guo-zheng ZHANG Ying-cai CHEN Gui-hui ZHANG Qi |
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| Affiliation: | YANG Qing,YANG Yang,LIU Jian-rong,PAN Guo-zheng,ZHANG Ying-cai,CHEN Gui-hui,ZHANG Qi(Department of Liver Transplantation,Third Affiliated Hospital,Sun Yat-sen University,Guangzhou 510630,China) |
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| Abstract: | 【Objective】To optimize the isolation and expansion protocol of human umbilical cord mesenchymal stem cells (hUC-MSCs). 【Methods】 Human UC-MSCs were isolated from 20 umbilical cords by combined enzyme digestion (n=12) or by sequential enzyme digestion (n=8). The derivation efficiency, primary cell culture period as well as cell yield were compared. The hUC-MSCs proliferation capacity was compared between complete low glucose-Dulbecco’s modified eagle medium (LG-DMEM) condition and LD-Mesen medium (a medium mixed with MesenPro RSTM (Invitrogen) and LG-DMEM) condition. The phenotypic characteristics of hUC-MSCs were determined by FACS and multi-lineage differentiation capacity was confirmed by induced adipogenesis and osteogenesis.【Results】 The hUC-MSCs derivation efficiency using combined enzymatic digestion was 100% (12/12), significantly higher than that of sequential enzymatic digestion [12.5% (1/8), P=1.03×10-4]. With combined enzymatic digestion, (1.30±0.14)×106 cells can be obtained during a mean primary cell culture period of (14.17±1.14) d. Moreover, 2×105 hUC-MSCs were cultured for 12 days, more cells can be obtained using LD-Mesen culture medium than using LG-DMEM culture medium [(14.86±0.08)×106 vs (5.08±0.08)×106, P=1.38×10-8]. hUC-MSCs express CD73, CD105, CD90, CD29, and CD44 surface markers, but do not express CD31, CD34, CD45 and HLA-DR. They can be also differentiated into osteoblasts and adipocytes in vitro. 【Conclusion】 hUC-MSCs can be efficiently derived by combined enzymatic digestion from Wharton’s Jelly and expanded by LD-Mesen expansion system, which is significantly superior to the conventional protocol. |
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| Keywords: | mesenchymal stem cells umbilical cord isolation |
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