慢性高原病患者骨髓间充质干细胞的体外扩增及鉴定

目的探讨慢性高原病（chronic mountain sickness，CMS）患者骨髓间充质干细胞（mesenchymal stem cell，MSCs）的体外培养方法、生物功能及分子生物学特征。方法采用密度梯度离心和贴壁筛选法对15例CMS患者（CMS组）和15例正常人（对照组）骨髓MSCs进行分离培养，于倒置显微镜下观察MSCs形态，用流式细胞仪行细胞表面抗原检测，检测MSCs生长情况，测定生长曲线。结果采用密度梯度离心法分离骨髓单个核细胞（BMMNC）后培养，骨髓MSC均分离培养成功，贴壁细胞呈梭形；两组骨髓MSCs均表达CD29、CD105和CD13，不表达CD45、CD4、CD8、CD14、CD3、CD34、HLA—DR和CD20。CMS组MSCs增殖能力高于对照组（P〈0．01），但传代率低于对照组（P〈0．05），对P3代细胞生长曲线进行观察发现CMS骨髓MSCs生长较对照组活跃。结论CMS患者骨髓MSCs在体外可有效分离培养，所培养扩增的细胞成分单一，CMS患者骨髓MSCs具有其特有的生物学特性。

INVITROAMPLIFICATION AND IDENTIFICATION OF BONE MARROW MESENCHYMAL STEM CELLS DERIVED FROM PATIENTS WITH CHRONIC MOUNTAIN SICKNESS

Objective To explore the in vitro culture approaches, biological functions and biological molecular characteristics of bone marrow mesenchymal stem cells （ MSCs ） derived from patients with chronic mountain sickness（CMS）. Methods The CMS was diagnosed by the Qinghai CMS Score established by the VI World Congress in 2004. MSCs were separated from bone marrow of 15 CMS patients（ CMS group） and 15 healthy adults（control group） after their agreement by density gradient centrifugation and adherent culture in vitro. MSCs were identified according to morphology and cell surface antigen profile. The morphological changes and the proliferation growth curves of MSCs were observed in primary and passage cultures. The surface makers were detected by flow Cytometry（FCM）. Results MSCs were successfully isolated from 15 CMS patients and controls. Adherent cells were spindle shape in morphology. It was found that MSCs expressed CD29, CDI05 and CD13 in flow Cytometry, but did not express CD45, CIM, CD8, CD14, CD3, CD34, HLA - DR and CD20. CMS - MSCs shared a similar morphologic characteristics and immune phenotypes with MSCs derived from normal bone marrow. The proliferation rate of MSCs in the CMS group was higher than that in the control group （P 〈0.01 ）, but the passage rate was lower than that in the control group （ P 〈 0.05 ）. It was found that the growth curve of the passage 3 cells that the growth of MSCs derived from CMS patients was more active than that of MSCs derived from the controls. Conclusion The bone marrow MSCs of CMS patients can be cultured purely and can extensively proliferate. And they possess specialbiological characteristics.