低氧抑制人骨髓间充质干细胞向成骨细胞分化  

Hypoxia Inhibiting the Osteogenic Differentiation of Adult Human Mesenchymal Stem Cells in vitro

作　　者：王庆德[1] 杨述华[1] 杨操[1] 胡勇[1] 赵继军[1] 潘晓华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院骨科,武汉430022

出　　处：《华中科技大学学报（医学版）》2008年第1期55-58,72,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No30750010)

摘　　要：目的探讨低氧对人间充质干细胞(hMSCs)分化为成骨细胞的影响,为骨组织工程学提供实验依据。方法根据培养氧浓度及培养液类型随机分为4组:正常氧组(n)(20%O2+DMEM-LG),低氧组(h)(1%O2+DMEM-LG),正常氧成骨诱导组(nos)(20%O2+条件培养液)及低氧成骨诱导组(hos)(1%O2+条件培养液)。观察细胞形态变化,检测碱性磷酸酶(ALP)活性,定量分析ALP、Ⅰ型胶原蛋白(COL1A2)、骨钙素(OC)及骨粘连蛋白(BSP)mRNA表达情况,钙结节使用茜素红染色。结果与nos组相比,n、h及hos组hMSCs生长形态改变不明显,并且不受条件培养液的影响。hos组hMSCs的ALP活性逐渐增高,但明显低于nos组,两者差异有统计学意义(P<0.01);培养4周后,与其它组相比,nos组的hMSCs可见到明显的染成红色的钙结节及钙盐沉积。定量RT-PCR检测,nos组hMSCs的ALP、OC、COL1A2及BSP mRNA表达量从7d时开始明显增加,ALP14d后达到峰值,随后活性开始降低,OC、COL1A2及BSP mRNA继续增高,hos组ALP及BSP mRNA14d后开始增加,但明显低于nos组(均P<0.05)。结论低氧环境在体外抑制hMSCs成骨细胞分化,延迟hMSCs成骨。Objective To investigate the effects of hypoxia on osteogenic differentiation of adult human mesenchymal stem cells （hMSCs） in vitro in order to provide experimental basis for the bone tissue engineering. Methods According to the oxygen concentrations and nutrient media, hMSCs were randomly divided 4 groups ： normoxic group （n） （20% O2 ＋DMEM-LG）, hypoxic group （h） （1% O2 ＋DMEM-LG）, normoxic osteoblast induction group （nos） （20% O2 ＋ conditioned medium）, hypoxic osteoblast induction group （hos） （1% O2 ＋conditioned medium）. The morphologic changes of hMSCs were observed under an inverted phasecontrast microscopy. The activity of alkaline phosphatase activity （ALP） was tested, the expression of ALP, collagen I A2 （COL1A2）, osteocalcin （OC） and bone sialoprotein （BSP） mRNA was detected by real-time RT-PCR, and the calcified nodules were stained by Alizarin red. Results Compared with nos group, there were no significant changes in morphology of hMSCs in n, h and hos groups. ALP activity of hMSCs in hos group was gradually increased, but less than in nos group （P〈0.01）. After culture for 4 weeks, red calcified nodules stained by Alizarin red were observed in nos group. Real-time RT-PCR revealed that in nos group, the expression of ALP, OC, COL1A2 and BSP mRNA in hMSCs was up-regulated at 7th day, and at 14th day, ALP reached the peak and then gradually reduced, but that of OC, COLIA2 and BSP mRNA was continuously increased. In hos group, the expression level of ALP, OC, COL1A2 and BSP mRNA in hMSCs at 14th day was increased, but obviously lower than in nos group （all P〈0.05）. Conclusion The osteogenic differentiation of hMSCs was sup- pressed by hypoxia in vitro.

关 键 词：间充质干细胞 低氧 成骨细胞 细胞培养 

分 类 号：Q813.11[生物学—生物工程] R318[医药卫生—生物医学工程]