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两种方法分离山羊骨髓间充质干细胞生长增殖能力的比较

发表时间:2016-06-15  浏览量:1610  下载量:866
全部作者: 吕玮,李燕梅,钟妮,张文霞,马晓芳,包广洁,康宏
作者单位: 兰州大学口腔医学研究所;西北民族大学口腔医学国家民委重点实验室
摘 要: 目的:比较percoll密度梯度离心法和全骨髓培养法体外培养山羊骨髓间充质干细胞(goat bone marrow-derived mesenchymal stem cells,gBMSCs)的纯度并评估其生物学特性,为后期实验摸清实验条件并提供大量细胞源。方法:无菌收取3~5月龄山羊股骨骨髓组织,分别采用percoll密度梯度离心法和全骨髓培养法体外分离gBMSCs,相差显微镜下每日观察细胞形态;取生长良好的P3代细胞,绘制细胞生长曲线;成纤维细胞集落形成单位(colony forming unit-fibroblast,CFU-F)实验检测其自我更新能力;流式细胞术检测细胞表面标志物表达。结果:两组中原代及传代细胞均呈漩涡状排列生长,两种分离方法获得的P3代细胞“S”形生长曲线形态相似,全骨髓培养法分离的细胞在成纤维细胞集落形成能力中表现出更高的自我更新能力,流式细胞术结果显示两者表面标志表达相似,其中CD34、CD44均呈阳性表达,而CD45呈阴性。结论:无论是密度梯度离心法还是全骨髓培养法,均可得到形态均一、具有自我更新潜力的gBMSCs. 但原代培养过程中密度梯度离心法提取的细胞增殖速度慢,到P3代时除细胞自我更新能力外其余细胞生物学特性无明显差异。
关 键 词: 口腔医学;组织工程;山羊;骨髓间充质干细胞;分离培养
Title: Isolation of goat bone marrow-derived mesenchymal stem cells: a comparison between two protocols on growth proliferation and biological characteristics
Author: LÜ Wei, LI Yanmei, ZHONG Ni, ZHANG Wenxia, MA Xiaofang1 BAO Guangjie, KANG Hong
Organization: Institute of Stomatology, Lanzhou University; Key Lab of Stomatology of State Ethnic Affairs Commission, Northwest University for Nationalities
Abstract: Objective: To investigate the two protocols of goat bone marrow-derived mesenchymal stem cells (gBMSCs) isolation: percoll gradient density separation protocols and holo-bone marrow adherence on purity and biological characteristics in vitro, and to provide large numbers of cells for subsequent experiments. Methods: Bone marrow was sterilely harvested from the thighbones of goat aged about 3-5 months, gBMSCs were isolated by both 2 protocols. The shapes of cells were observed by inverted phase contrast microscopes. The colony forming unit-fibroblast (CFU-F) assay was performed to test those self-renewing. Moreover, the growth curve of passage P3 cells was drawn and surface marker expression of P3 cells was investigated by flow cytometry (FCM). Results: The primary and passaged cells were spindle-shaped and grew in colonies. The growth curves of cultured gBMSCs by both two protocols was like “S” shape. The results of CFU-F assay demonstrated that cells harvestd by holo-bone marrow adherence possessed higher self-renewal capacities. Two kinds of cells were uniform in surface marker expression. Both CD34 and CD44 were positive expression and CD45 was negative expression. Conclusion: Both methods can obtain homogenous gBMSCs with self-renewal capacities. Though cells at P0 retrieved with the percoll protocol had lower proliferation when compared with the holo-bone marrow adherence, there was no significant difference between the two protocols at P3 except self-renewal capacity.
Key words: stomatology; tissue engineering; goat; bone marrow-derived mesenchymal stem cells; separation of culture
发表期数: 2016年6月第11期
引用格式: 吕玮,李燕梅,钟妮,等. 两种方法分离山羊骨髓间充质干细胞生长增殖能力的比较[J]. 中国科技论文在线精品论文,2016,9(11):1121-1129.
 
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