骨髓间充质干细胞成纤维样集落的培养方法及多向分化潜能  

Fibroblast colony formation by primary culture of mesenchymal stem cells and its multi-differentiation potentials

作　　者：刘岐焕[1] 程范军[2] 高清平[1] 陈龙[2] 唐俊明[3] 王家宁[3] 

机构地区：[1]武汉大学医学院人民医院血液科,湖北省武汉市430060 [2]郧阳医学院附属东风总医院血液科,湖北省十堰市442000 [3]郧阳医学院附属人民医院临床医学研究所,湖北省十堰市442000

出　　处：《中国临床康复》2006年第13期35-37,i0002,共4页Chinese Journal of Clinical Rehabilitation

基　　金：湖北省自然科学基金(2005ABA081)~~

摘　　要：目的:建立原代小鼠骨髓间充质干细胞成纤维样集落的培养方法,了解间充质干细胞的培养特性及其多向分化潜能。方法:实验于2004-10/2005-10在郧阳医学院附属人民医院临床医学研究所完成。①间充质干细胞的分离:选取SPF级2月龄昆明种小鼠30只,无菌条件下抽取双侧股骨、胫骨骨髓,制成骨髓单细胞悬液,计数有核细胞的密度。②血清浓度对成纤维样集落的影响:调整有核细胞密度为5×108L-1,分别接种于体积分数为0.02,0.05,0.1,0.15,0.2,0.25的胎牛血清-DMEM培养基中,每种血清浓度各4孔,1mL/孔,24h后换液。随后每72h换液,连续观察2周。③有核细胞密度对成纤维样集落的影响:分别调整有核细胞至106L-1,107L-1,2.5×108L-1,5×108L-1,109L-1,分别接种于12孔板中,每种密度4孔,24h后换液。随后每72h换液,连续观察2周,瑞氏吉姆萨染色后计数成纤维样集落的个数。④成骨与成脂肪诱导:调整有核细胞密度在5×108L-1,接种于预包被鼠尾胶的12孔板中培养。24h换液,去除未贴壁的细胞,以后每48h换液1次,换液后加入成骨诱导液、成脂肪诱导液。观察各血清浓度下细胞生长情况,消化培养板中的成纤维样集落流式细胞仪检测CD34,CD133,CD90,CD105。VonKossa和油红O法染色鉴定间充质干细胞成骨与成脂肪诱导情况。结果:①不同血清浓度对成纤维样集落形成的影响:胎牛血清-DMEM培养液体积分数为0.02,0.05时,细胞可保持成纤维样,但增殖较慢;体积分数为0.15,0.2,0.25时,细胞增殖较快,但细胞易分化;体积分数为0.1时,细胞可基本保持成纤维样,且增殖速度适中。②有核细胞不同接种密度对成纤维样集落形成的影响:有核细胞接种密度为106L-1,107L-1时,每孔成纤维样集落数为0～1个。接种密度高于109L-1时,每孔成纤维样集落之间出现交叉或融合。接种密度分别为2.5×108L-1,5×108L-1,109L-1时,10d左右可见典型的成纤维样集落。�AIM： To establish the primary culture of bone marrow-derived mesenchymal stem cells （MSCs） fibroblast colony in mice, and investigate the culture characteristics and multi-differentiation potentials of MSCs. METHODS： The experiment was conducted in the Institute of Clinical Medicine, Affiliated People＇s Hospital, Yunyang Medical College between October 2004 and October 2005. ①The isolation of MSCs： Thirsty Kunming mice of SPF Grade, aged 2 months were selected to extract the bone marrows from femur and tibia in the sterile condition and prepare the monocellular suspension. Then the densities of bone marrow nucleated cells （BMNCs） were counted.②Effect of serum concentrations on fibroblast colony： With the adjusted density of 5×10^8 L^-1, BMNCs were inoculated onto the DMEM medium with the fetal bovine serum （FBS） volumes of 0.02, 0,05, 0,1, 0.15, 0.2 and 0.25, Each serum concentration was consisted of 4 holes, 1 mL per hole. The culture medium were replaced 24 hours later, and then replaced every 72 hours for the observation of two weeks. ③Effect of BMNCs＇ density on fibroblast colony： With the adjusted densities of 10^t L^-1,10^7 L^-1,2.5×10^8 L^-1,5×10^8 L^-1 and 10^9 L^-1, BMNCs were respectively inoculated onto the 12-hole plate with 4 holes of same density. The medium was replaced 24 hours later, and then continued to replace every 72 hours for the observation of two weeks. The number of fibroblast colony was counted after Giemsa staining was performed. ④Inductions by adipocyte and bone： With the adjusted density of 5×10^8 L^-1, BMNCs were inoculated and cultured on the 12-hole plate which was coated by rat-tail collagen. After 24 hours, the medium was replaced to wipe off the cells, which was not adhesive to the cell wall, and then continued to replace every 48 hours with the additions of bone and adipocyte induction liquids. The growths of MSCs were observed at different concentrations of serum. The colony-forming unit-fibroblast （CFU-F） was digested in the cult

关 键 词：干细胞移植 骨髓细胞 间质细胞 集落形成单位测定 细胞分化 

分 类 号：R394.2[医药卫生—医学遗传学]