人骨髓间充质干细胞亚群ZHJ-MAPCs特征表型及细胞遗传学分析

Phenotype and genetic analysis of ZHJ-MAPCs of human mesenchymal stem cells subgroup

目的:对经过CD45,血型糖蛋白-A免疫微磁珠体外负分选纯化并传代培养的ZHJ-MAPCs进行体外部分细胞学鉴定,了解其特异性标志物表达及增殖、传代过程中的遗传学稳定性。方法:实验于2004-01/12在珠江医院中心试验室完成。①原代冻存的ZHJ-MAPCs经复苏培养、传代。②利用流式细胞仪对其进行CD29,CD44,CD34和HLA-DR流式细胞分析,以对照管作为空白定标,计算10000个细胞,记录标本的阳性细胞百分率。③分别在传代至第6代和第13代时进行细胞核型分析了解细胞核型变化。④细胞计数及细胞活力检测方法:细胞数/mL=四个大方格总数/4×104×稀释倍数。细胞存活率=5g/L锥虫蓝染色阴性细胞/100个细胞/高倍镜×100。结果:①流式细胞仪检测结果:流式细胞仪检测ZHJ-MAPCs中CD29阳性表达细胞比率为99.2%,CD44阳性细胞比率为98.3%,CD34阳性细胞比率为1.2%,HLA-DR阳性细胞比率为5.3%。②细胞核型分析结果:ZHJ-MAPCs在增殖传代过程中表现出非正常二倍体特征,染色体干系数目在第6,13代均为55～58条,占80%,染色体数介于超二倍体和亚三倍体之间,镜下见细胞均发生染色体变异,且变异形态多样。结论:①ZHJ-MAPCs具有骨髓间充质干细胞的特征及较高的纯度。②ZHJ-MAPCs在增殖传代过程中表现出非正常二倍体特征,染色体数介于超二倍体和亚三倍体之间,染色体变异形态多样。

AIM： To understand the specific phenotypes and genetic stability in proliferation and passage after cytology evaluation in vitro continuously cultured ZHJ-MAPCs purified by CD45 and glycophorin-A （GlyA） negative magnetic activated cell sorting. METHODS： The experiment was performed at the Central Laboratory of Zhujiang Hospital from January to December 2004. ①Original preservation ZHJ-MAPCs through revivification culture, passage. ②CD29, CD44,CD34 and HLA-DR flow cytometry was conducted with flow cytometer. Blank calibration was considered as control. 10 000 cells were calculated. Percentage of positive cells of samples was recorded. ③Cells karyotype analysis was performed at the 6^th and 13^th passage to analyze karyotype change. ④Detection of cell counting and cell activity： number of cells/mL was equaled to sum of the four large checks/4 ×10^4 ×dilution power. Survival rate of cells was equaled to 5 g/L Trypan blue staining negative cells/100 cells/high-pewer microscope×100. RESULTS：①Detection result of flow cytometer＇. CD29 positive ratio in ZHJ-MAPCs was 99.2%, with CD44 positive ratio 98.3%, CD34 positive ratio 1.2% and HLA-DR positive ratio 5.3%. ② Analytic result of karyotype： Continuously cultured ZHJ-MAPCs did not show normal diploids. The number of chromosome were 55-58 at generation 6 and 13, accounting for 80% of all chromosomes. Number of chromatosome was between that of hyperdiploid and sub-triploid. Chromosomal shapes were also diversified under microscope. CONCLUSION： ① ZHJ-MAPCs are highly purified cell with the phenotype characteristics of mesenchymal stem cells （MSCs）. ②After continuous culture, ZHJ-MAPCs do not have normal diploids, and number of chromatosome was between that of hyperdiploid and sub-triploid. Chromosomes have diversified shapes under microscope.