骨保护素的克隆及对骨髓间充质干细胞分化的影响  

The clone of OPG and its effects on the differentiation of bome morrow stem cells

作　　者：陈富[1] 徐希彦[1] 柯珍勇[1] 尹良军[1] 郭常辉[1] 王雪燕[1] 林春阳[1] 邓忠良[1] 

机构地区：[1]重庆医科大学附属第二医院骨科,重庆400010

出　　处：《重庆医科大学学报》2009年第5期532-536,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金(30672121);重庆市教委科研课题(渝教科2002-27)。

摘　　要：目的:克隆人骨保护素(Osteoprotegerin,OPG)基因并构建其重组腺病毒,观察其对BMSCs分化的影响.方法:RT-PCR方法从人肝克隆OPG基因全长cDNA,利用穿梭质粒将OPG基因重组获得复制缺陷型重组腺病毒AdOPG;分离培养兔骨髓间充质干细胞,使用AdOPG感染兔BMSCs,以RT-PCR及Western Blot的方法鉴定BMSCs对OPG的表达。比色测定和细胞碱性磷酸酶染色法观察AdOPG感染BMSCs后5d时碱性磷酸酶(ALP)的表达。结果:克隆获得人OPG基因,测序与Gene bank一致,构建的重组腺病毒AdOPG滴度达109efu/ml,AdOPG感染BMSCs5d后ALP活性值为(21024±507)IU,AdGFP对照组为(3079±89)IU,空白对照组为(2156±78)IU。结论:骨保护素有利于骨髓间充质干细胞向成骨细胞方向分化。Objective： To clone the OPG gene of human being and construct its recombinant adenovirus and then observe its effects on the differentiation of bone marrow stem cells （ BMSCs ）. Methods ： The cDNAs of human OPG in hepatic tissue were obtained by using RT-PCR method.The cDNAs then were recombined with competent homologous cells to form the AdOPG and the shuttle vector was used during this stage. The rabbit BMSCs were isolated and cultured in vitro, and were infected with AdOPG. The OPG expression of BMSCs was identified by RT-PCR and Western blot. The expression of ALP was detected by chromatometry and ALP dyeing five days after the infection. Results： The OPG gene of human being was successfully cloned and its sequence consistent with the Gene bank. The titre of constructed AdOPG can reach 109 efu/ml. The ALP activity of BMSCs was （21 024 ± 507） IU five days after the infection of AdOPG, whereas the value was （3 079 ± 89） IU in the control group and （2 156 ± 78） IU in the blank control group. Conclusion： The OPG can induce the osetogeinc differentiation of BMSCs.

关 键 词：腺病毒 人骨保护素 骨髓间充质干细胞 

分 类 号：R336[医药卫生—人体生理学]