实验兔骨髓间充质干细胞的分离、培养及鉴定

发布时间：2018-06-01 03:26

本文选题：骨髓间充质干细胞 + 细胞培养　；参考：《基因组学与应用生物学》2017年01期

【摘要】：探讨建立兔骨髓间质干细胞(BMSCs)的体外分离纯化、扩增和鉴定的方法,为BMSCs的进一步诱导分化和应用奠定基础。首先抽取兔髂骨骨髓,采用Percoll密度梯度离心法得到骨髓单个核细胞,接种后形成单层贴壁细胞,经胰蛋白酶消化后传代培养扩增,倒置相差显微镜观察细胞生长状态,细胞免疫组化检测CD73、CD34。结果表明,成功建立了兔BMSCs体外分离及培养扩增的方法,发现BMSCs表现贴壁生长,P0代时呈集落生长,细胞呈梭形,传代后细胞增殖速度加快,形态开始多样化;细胞免疫组化显示BMSCs表达CD73,未表达CD34,反复传代后CD73表达效率增高。上述研究表明,Percoll密度梯度离心联合骨髓贴壁法,能有效分离、纯化和扩增BMSCs,提取的细胞具有BMSCs的生长特性和抗原表型,其中P3~P6代细胞增殖能力强,可用于进一步的研究工作。
[Abstract]:To establish the method of isolation, purification, amplification and identification of rabbit bone marrow mesenchymal stem cells (BMSCs) in vitro, and to lay a foundation for further induction, differentiation and application of BMSCs. Bone marrow mononuclear cells were obtained from rabbit iliac bone marrow by Percoll density gradient centrifugation. Monolayer adherent cells were formed after inoculation. The monolayer adherent cells were cultured and amplified by trypsin digestion. The growth state of the cells was observed by inverted phase contrast microscope. CD73 and CD34 were detected by immunohistochemistry. The results showed that the method of isolation and culture of rabbit BMSCs in vitro was successfully established. It was found that BMSCs showed colony growth at P0 generation and fusiform cells. After passage, the proliferation rate of rabbit BMSCs increased and the morphology began to diversify. Immunohistochemistry showed that BMSCs expressed CD73, but not CD34. After repeated passage, the efficiency of CD73 expression was increased. These results showed that Percoll density gradient centrifugation combined with bone marrow adherent method could effectively isolate, purify and amplify BMSCs. The extracted cells had the growth characteristics and antigenic phenotypes of BMSCs, and the P3~P6 generation cells had strong proliferative ability, which could be used for further research.
【作者单位】：广州中医药大学深圳临床医学院;深圳北京大学香港科技大学医学中心生物医学研究所;广州中医药大学深圳医院;
【基金】：深圳市科技计划(卫生系统)科研项目(项目编号:201302124) 深圳市福田区卫生公益性科研项目(项目编号FTWS201318)共同资助
【分类号】：R329.2

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