Culture and identification of rhesus monkey bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.29.006

Abstract

Abstract:

BACKGROUND: Studies on rhesus monkey bone marrow mesenchymal stem cells (BMSCs) are limited, and the differentiation 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 the whole bone marrow adherence methods. BMSCs at passages 2 and 7 were taken to draw cell growth curves by MTS method. Flow cytometry was used to detect apoptosis of passage 3 BMSCs and to detect expression of cell surface markers, CD29, CD34, CD45, CD90, CD147, in passage 4 BMSCs. Osteogenic and adipogenic abilities of passage 3 BMSCs were also detected.
RESULTS AND CONCLUSION: The BMSCs at passages 2 and 7 were incubated at first 2 days, logarithmically grew at 3-7 days, and then proliferated slowly at 8 days. The BMSCs at passage 2 showed stronger proliferation than those at passage 7. At passage 3, BMSCs were negative for CD34 and CD45, but positive for CD90, CD29 and CD147. Results from the flow cytometry showed that there were 73.83% living cells, 21.76% dead cells, and 4.27% apoptotic cells. BMSCs could differentiate into osteoblasts and adipocytes under osteogenic and adipogenic induction, respectively. To conclude, BMSCs from the rhesus monkey have strong proliferation and multilineage differentiation abilities in vitro.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Mesenchymal Stem Cells, Macaca mulatta, Cell Culture Techniques, Tissue Engineering

CLC Number:

R394.2

Dong Jing-jing, Hu Zhu-lin, Li Yan, Sun Xiao-mei. Culture and identification of rhesus monkey bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(29): 4623-4628.

Figures/Tables 1

2.1 恒河猴骨髓间充质干细胞的形态学观察骨髓间充质干细胞接种于25 cm2培养瓶后，细胞呈圆形，大小不一，悬浮于完全培养液中；24 h后部分细胞开始贴壁，贴壁细胞呈梭形；通过半量换液去除部分未贴壁的杂质细胞，48 h后可见短梭形、星形细胞分散贴壁生长；原代细胞生长缓慢，七八天可见漩涡状排列的细胞集落，伸出长短不一、粗细不均的突起，培养10 d细胞集落生长(图1)，14 d细胞排列紧密，逐渐融合成片达到80%-90%融合，进行传代培养。细胞消化传代后，传代细胞24 h完全贴壁生长。细胞呈梭形，生长旺盛，六七天可传代1次(图2)，可稳定连续传代10代以上，细胞形态未见明显变化，6-10代细胞生长速度略微减慢，八九天传代1次。 2.2 恒河猴骨髓间充质干细胞的生长曲线第2，7代骨髓间充质干细胞在培养第一二天为细胞潜伏期，第3-7天生长曲线基本为线性曲线，表明第3-7天是细胞的对数生长期，第8天后曲线逐渐变得平缓，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(图3)。 2.3 恒河猴骨髓间充质干细胞的凋亡检测流式细胞仪细胞凋亡分析结果显示，73.83%为活细胞，21.76%为死亡细胞，4.27%为凋亡细胞，见图4，表明骨髓间充质干细胞具有良好的生物学活性。 2.4 恒河猴骨髓间充质干细胞表面标记物的表达流式细胞仪检测结果表明，第4代骨髓间充质干细胞表面标记物CD90、CD29、CD147呈阳性表达，阳性率分别为98.8%，96.4%，89.7%；CD34、CD45呈阴性表达，阳性率均为0(图5)。重复3次结果一致。 2.5 恒河猴骨髓间充质干细胞诱导分化鉴定成骨细胞诱导：成骨诱导21 d后茜素红染色，倒置显微镜下显示实验组有大量橘红色结节形成，对照组显示为阴性(图6)。成脂细胞诱导：成脂诱导24 d后油红O染色，倒置显微镜下显示实验组胞浆内出现大量红色脂滴，形态大小不一，成串排列或围绕细胞核分布；对照组染色后细胞内未见红染脂滴(图6)。"

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