恒河猴骨髓间充质干细胞的培养与鉴定

Culture and identification of rhesus monkey bone marrow mesenchymal stem cells

背景:关于恒河猴骨髓间充质干细胞的研究结果很有限,来自幼年恒河猴骨髓间充质干细胞的分化潜力仍然未知。目的:探讨体外分离培养的恒河猴骨髓间充质干细胞生物学特性,对其生长曲线和表型进行初步鉴定。方法:采用密度梯度离心法和全骨髓贴壁培养法提取培养幼年恒河猴骨髓间充质干细胞,利用MTS法绘制第2,7代骨髓间充质干细胞生长曲线,利用流式细胞仪分析法检测第3代骨髓间充质干细胞凋亡及第4代骨髓间充质干细胞表面标志物CD29、CD34、CD45、CD90、CD147表达,检测第3代骨髓间充质干细胞的成骨及成脂分化能力。结果与结论:①第2,7代骨髓间充质干细胞在培养第一二天为潜伏期,第3-7天为对数生长期,第8天后细胞增殖逐渐减慢,进入平台期,第7代细胞增殖能力弱于第2代;②骨髓间充质干细胞表面标志物CD90、CD29、CD147呈阳性表达,CD34、CD45呈阴性表达;③流式细胞仪细胞凋亡检测结果显示,73.83%为活细胞,21.76%为死亡细胞,4.27%为凋亡细胞;④骨髓间充质干细胞可诱导分化为成骨细胞与成脂细胞;⑤结果表明,分离培养的恒河猴骨髓间充质干细胞生长活跃,有较强的增殖及多向分化能力。

BACKGROUND:Studies on rhesus monkey bone marrow mesenchymal stem cells(BMSCs)are limited,and thedifferentiation potential of juvenile rhesus monkey BMSCs remains unknown.OBJECTIVE:To investigate the biological characteristics of rhesus monkey BMSCs isolated and cultured in vitro,and to identify the growth curve and phenotype of BMSCs.METHODS:BMSCs from juvenile rhesus monkeys were isolated and cultured by density gradient centrifugation and thewhole bone marrow adherence methods.BMSCs at passages2and7were taken to draw cell growth curves by MTSmethod.Flow cytometry was used to detect apoptosis of passage3BMSCs and to detect expression of cell surfacemarkers,CD29,CD34,CD45,CD90,CD147,in passage4BMSCs.Osteogenic and adipogenic abilities of passage3BMSCs were also detected.RESULTS AND CONCLUSION:The BMSCs at passages2and7were incubated at first2days,logarithmically grew at3-7days,and then proliferated slowly at8days.The BMSCs at passage2showed stronger proliferation than those atpassage7.At passage3,BMSCs were negative for CD34and CD45,but positive for CD90,CD29and CD147.Resultsfrom the flow cytometry showed that there were73.83%living cells,21.76%dead cells,and4.27%apoptotic cells.BMSCs could differentiate into osteoblasts and adipocytes under osteogenic and adipogenic induction,respectively.Toconclude,BMSCs from the rhesus monkey have strong proliferation and multilineage differentiation abilities in vitro.