[人骨髓间充质干细胞的体外缺氧培养及其在成人中的生物学特性]

[In vitro hypoxic culture of human marrow mesenchymal stem cells and their biological features in adults].

作者信息

Wang Qingde, Yang Shuhua, Yang Cao

机构信息

Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan Hubei 430022, PR China.

出版信息

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2007 Oct;21(10):1128-32.

PMID:17990784

Abstract

OBJECTIVE

To establish a model of the human marrow mesenchymal stem cells (hMSCs) cultured under the hypoxic condition in adults and to investigate the biological features of MSCs under hypoxia.

METHODS

The bone marrow was obtained by aspiration at the posterior superior iliac spine in 3 healthy adult subjects. hMSCs were isolated by the gradient centrifugation and were cultured in the DMEM-LG that contained 20% fetal bovine serum. The serial subcultivation was performed 10-14 days later. The second passage of the hMSCs were taken, and they were divided into the following 4 groups according to the oxygen concentrations and the medium types: the normoxic group (20% O2, DMEM-LG, Group A), the hypoxic group (1% O2, DMEM-LG, Group B), the normoxic osteoblast induction group (20% O2, conditioned medium, Group C), and the hypoxic osteoblast induction group (1% O2, conditioned medium, Group D). The biological features of the cultured hMSCs under hypoxia were assessed by the cell count, the MTT method, the colony forming unit-fibroblast, the real-time RT-PCR, and the alkaline phosphatase (ALP) activity, and the alizarin red staining.

RESULTS

The hMSCs cultured in the Group B and Group D had a significantly higher proliferation rate than those in the Group A (P < 0.01), and the culture effect was not influenced by the medium type. The hMSCs in the Group B had a significantly higher level of the colony-forming unit capability than the hMSCs cultured in the Group A (P < 0.01). After the induction, hMSCs in the Group B had a decreased number of the osteoblasts than hMSCs in the Group C. The hMSCs in the Group D had a gradually-increased activity of ALP, which was significantly lower than that in the Group C (P < 0.01). The RT-PCR examination revealed that ALP, osteocalcin, and mRNA expressions of collagen type I and osteonectin in the Group C significantly increased (P < 0.01). By comparison among the 3 groups, after the 4-week culture the obvious calcium salt deposit and the red-stained calcium nodus could be observed.

CONCLUSION

Hypoxia can promote the proliferation rate of hMSCs, enhance the colony-forming ability and inhibit the differentiation of the osteoblasts.

摘要

目的

建立成人骨髓间充质干细胞（hMSCs）低氧培养模型，探讨低氧条件下MSCs的生物学特性。

方法

抽取3例健康成人志愿者后上棘骨髓。采用梯度离心法分离hMSCs，接种于含20%胎牛血清的低糖DMEM培养基中培养，10～14 d后传代。取第2代hMSCs，根据氧浓度和培养基种类分为4组：常氧组（20% O2，低糖DMEM培养基，A组）、低氧组（1% O2，低糖DMEM培养基，B组）、常氧成骨诱导组（20% O2，条件培养基，C组）、低氧成骨诱导组（1% O2，条件培养基，D组）。采用细胞计数、MTT法、集落形成单位-成纤维细胞、实时荧光定量RT-PCR、碱性磷酸酶（ALP）活性测定及茜素红染色等方法，观察低氧条件下培养的hMSCs生物学特性。

结果

B组和D组hMSCs增殖率明显高于A组（P < 0.01），培养基种类对培养效果无影响。B组hMSCs集落形成能力明显高于A组（P < 0.01）。诱导后，B组hMSCs分化为成骨细胞的数量少于C组。D组hMSCs的ALP活性逐渐升高，但明显低于C组（P < 0.01）。RT-PCR检测显示，C组ALP、骨钙素、Ⅰ型胶原和骨连接蛋白mRNA表达明显增加（P < 0.01）。3组比较，培养4周后可见明显的钙盐沉积和红色钙结节。