成人骨髓间充质干细胞的体外培养及生物学特性

In vitro culture and biological characteristics of adult bone marrow mesenchymal stem cells

目的:观察成人骨髓间充质干细胞体外分离培养方法及其生物学特性,为骨髓间充质干细胞的临床应用奠定技术基础。方法:实验于2005-12在吉林省四平市中心医院中心实验室进行。实验材料:人骨髓间充质干细胞,Ficoll淋巴细胞分离液,胎牛血清,牛血清白蛋白,α-MEM培养基,胰蛋白酶,FITC标记的CD44和CD90。实验方法:①人骨髓间充质干细胞的分离与培养:采集成人骨髓,密度梯度法分离单个核细胞,条件培养基培养和扩增,倒置光学显微镜下观察细胞形态及生长情况。②人骨髓间充质干细胞生长曲线的测定:取生长良好的P1,P5,P10细胞,用胰酶消化,接种于24孔板培养,每天各取3孔消化计数细胞,取平均值,连续培养7d,绘制生长曲线。③人骨髓间充质干细胞表面标志的测定:取生长良好P5细胞进行免疫荧光染色鉴定(CD44,CD90并设同型对照)。④人骨髓间充质干细胞的冻存及复苏:收集生长良好细胞,计数细胞密度及冻前存活率。将细胞加入到细胞冻存液中液氮冷冻保存。30d后复苏细胞,每日倒置光学显微镜下观察细胞生长状况。结果:①倒置显微镜下观察细胞形态:倒置显微镜下观察细胞呈纤维形,漩涡状生长。②人骨髓间充质干细胞的生长曲线分析:传代的细胞生长较原代要快,从第10代开始细胞生长开始出现缓慢征象。③细胞表面标记检测结果:免疫荧光染色后细胞膜着色明显,CD44,CD90阳性率均大于90%。④细胞冻存及复苏生长特性分析:椎虫蓝计数细胞存活率90%以上,增殖能力强,和未冻存过的传代细胞具有同样的生长特性。结论:应用密度梯度法分离法能获得较高纯度的骨髓间充质干细胞,能在体外长期培养,在传代培养5代内生长旺盛且生物学特性稳定。

AIM： To observe adult bone marrow mesenchymal stem cells （hBMSCs）isolating and cultivating method and biological character in vitro for clinic application. METHODS： The experiment was performed at the Central Laboratory, Siping Central Hospital in December 2005. Experimental materials included hBMSCs, Ficoll, fetal calf serum （FCS）, bovine serum albumin （BSA）, （x-MEM, trypsin, CD44 and CD90 marked by FITC.①lsolating and cultivating hBMSCs： Bone marrow was obtained from healthy adult. Mononuclear cells ware isolated and purified by density and gradient centrifuge, cultivated by culture medium. After successful proliferation, the growth morphology and growth were observed under inverted light microscope. ②Drawing the growth curve of hBMSCs： The cells （P1, P5, P10） were assimilated by trypsin and cultivated in 24-well plate. Three bores were assimilated every day for 7 days, and then growth curve with average was drawn. ③Identifying the cell-surface marker： The well-growth P5 cells were identified by immunofluorescence staining （CD44 and CD90 as homeotype control）. ④Freeze conserve and anabiosis of hBMSCs： The well-growth cells were conserved with liquid in fluid nitrogen after taking count of cell density and livability. 30 days later, cell anabiosis was conducted, and then cell growth was observed every day with inverted light microscope. RESULTS： ①Observing the morphology of hBMSCs with inverted microscope： Cells showed fiber figure and swirl growth. ②Analysis of the growth curve： Passage cells grew faster than original generation. Cells growth appeared slowly from the tenth generation. ③The result of the cell-surface markers： Cell membranes were colored up evidently after immunofluorescence staining, and CD44 and CD90 positive rate were above 90%. ④Analysis of freeze conserve and anabiosis of hBMSCs： The cells livability was above 90%, and increasing capacity was as strong as un-conserved passage cells. CONCLUSION： Better quantity and purity hBMSCs are obtained by density and gradient separate method, hBMSCs can be cuttured long period in vitro and develop bloom within five passages with stable biological characteristics.