人骨髓间充质干细胞定向成骨诱导中碱性磷酸酶对其成骨能力的影响  

Effect of alkaline phosphatase on ossification capacity of human marrow-derived mesenchymal stem cells （hBMSCs） during hBMSCs differentiating into osteoblasts

作　　者：柳向东[1] 唐朝晖 白祥军 崔磊[3] 曹谊林[3] 

机构地区：[1]华中科技大学同济医学院附属协和医院整形外科,武汉430030 [2]附属同济医院创伤外科 [3]上海交通大学医学院附属第九人民医院整形外科

出　　处：《中华创伤杂志》2009年第10期945-948,共4页Chinese Journal of Trauma

基　　金：基金项目：国家高技术研究发展计划资助项目（2002AA205011）;湖北省自然科学基金资助项目（2007ABA168）

摘　　要：目的探讨窟向诱导人骨髓间充质干细胞（hBMSCs）分化为成骨细胞过程中，碱性磷酸酶（ALP）对hBMSCs成骨能力的影响。方法抽取骨髓，梯度离心法分离单个核细胞，在DMEM条件培养基诱导hBMSCs定向分化为成骨细胞；相差显微镜观察成骨细胞形态，MTF法检测成骨细胞增殖，测定成骨细胞ALP分泌量及成骨细胞胞内ALP含量，RT—PCR检测成骨细胞ALP mRNA的表达。结果梯度密度离心和黏附贴壁法联合使用可得到均一的hBMSCs；体外定向诱导过程中，成骨细胞ALP含量和ALP mRNA的表达在定向诱导10d左右开始显著升高，成骨细胞的增殖能力明显增强。随着传代次数的增加，成骨细胞的增殖能力和碱性磷酸酶分泌量以及ALP mRNA的表达呈下降趋势，与BM—Purple染色结果一致。结论hBMSCs经定向诱导后可以分化为成骨细胞，ALP含量的变化能够显著影响hBMSCs诱导成骨的能力。Objective To investigate the potential role of alkaline phosphatase （ALPase） in ossification capacity of human marrow-derived mesenchymal stem cells （hBMSCs） during hBMSCs differentiating into osteoblasts. Methods hBMSCs were separated by gradient centrifugation on Percoll, cultured with ordinary culture medium and induced by conditional culture medium. The morphous of the hBMSCs cultured with conditional culture medium was examined by contrast phase microscope. MTT assay was used to detect proliferation of the osteoblasts and ALPase contents in the osteoblasts. RT-PCR was employed to detect ALP mRNA expression of the osteoblasts. Results Homogeneous hBMSCs could be obtained by using gradient centrifugation and attachment method. ALP contents and ALP mRNA expressions of the osteoblats were increased markedly 10 days after in vitro induction, with significant augmentation of the osteoblasts. With increase of generation, there showed a tendency of decrease on proliferation capacity of the osteoblasts, the content of ALPase and ALP mRNA expression in the osteoblasts, which was in accordance with the results of BM-Purple staining. Conclusions hBMSCs can be induced to differentiate into osteoblasts in conditional culture medium in vitro. Osteogenesis of hBMSCs can be influenced obviously by content change of ALPase.

关 键 词：间充质干细胞 细胞分化 成骨细胞 碱性磷酸酶 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R446.112[医药卫生—基础医学]