AdCMV-BMP-7促进兔骨髓基质细胞的成骨分化  

Osteoblastic differentiation of rabbit bone marrow stromal cells by recombinant adenovirus cytomegalovirus bone morphogenetic protein-7

作　　者：施伟伟[1] 叶晓健[1] 许国华[1] 顾韬[1] 何海龙[1] 张桦[1] 

机构地区：[1]解放军第二军医大学长征医院骨科

出　　处：《中国组织工程研究与临床康复》2008年第25期4860-4864,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金重点项目(30400451)~~

摘　　要：目的:研究表明骨形态发生蛋白7可促进软骨的再生和重建。实验拟证实人骨形态发生蛋白7可促进兔骨髓基质细胞分化为成骨细胞。方法:实验于2005-10/2006-03在解放军第二军医大学长征医院完成。①实验材料:pAdTrack-CMV,pAdEasy-1及pGEM-T-BMP-7质粒均由许国华博士惠赠;大肠杆菌TG1、DH5a、BJ5183及包装细胞株293为本实验室保存;成年雄性新西兰大白兔2只购于解放军第二军医大学动物中心。②实验过程及评估:构建重组腺病毒表达载体AdCMV-BMP-7:首先将骨形态发生蛋白7序列插入到穿梭载体pAdTrack中,得到重组腺病毒pAdCMV-BMP-7;传代培养至第3代兔骨髓基质细胞,加入不同滴度值的AdCMV-BMP-7培养7d,以感染腺病毒空载体和未感染AdCMV-BMP-7的兔骨髓基质细胞作为阴性对照,观察碱性磷酸酶含量与时间及剂量的关系。结果:①重组AdCMV-BMP-7的构建:采用的AdEasyTM-1载体系统含有报告基因绿色荧光蛋白,筛选时间大为缩短,得到大约34kb大小的AdCMV-BMP-7。②AdCMV-BMP-7促进兔骨髓基质细胞分化结果:感染AdCMV-BMP-7后的骨髓基质细胞合成碱性磷酸酶明显增加且呈时间及剂量依赖性,而骨髓基质细胞感染腺病毒空载体对碱性磷酸酶合成无影响,提示骨髓基质细胞在单一的骨形态发生蛋白7作用下即可分化为成骨细胞。结论:实验结果提示重组腺病毒表达载体AdCMV-BMP-7能够促进骨髓基质细胞向成骨细胞方向分化,且成时间依赖性及剂量依赖性。AIM： Bone morphogenetic protein-7 （BMP-7） can improve the regeneration and reconstruction of the cartilage. This study was aimed to confirm the effect of human BMP-7 on stimulating rabbit bone marrow stromal cells to differentiate to osteoclasts. METHODS： The experiment was completed in the Affiliated Changzheng Hospital of the Second Military Medical University of Chinese PLA from October 2005 to March 2006. Materials： Plasmid pAdTrack-CMV, pAdEasy-1 and pGEM-T-BMP-7 were all donated by Doctor Xu Guo-hua; E coli TG1, DH5a, BJ5183 and package cell strain 293 were stored in this laboratory; Two adult male New Zealand white rabbits were bought from the animal center of the Second Military Medical University of Chinese PLA. Construction of recombinant adenovirus AdCMV-BMP-7： Firstly, the sequence of BMP-7 was inserted into shuttle vector pAdTrack to construct AdCMV-BMP-7; Secondly, rabbit bone marrow stromal cells at the third passage were added with AdCMV-BMP-7 of different multiplicity of infection, and cultured for 7 days, while the marrow stromal cells infected with blank adenovirus vector and the uninfected marrow stromal cells were taken as controls. Then the correlation of alkaline phosphatase （ALP） with time and dosage was observed. RESULTS： AdCMV-BMP-7 was constructed with AdEasy-lTM vector system carrying report gene green fluorescent protein, which could shorten screen time obviously. AdCMV-BMP-7 of about 34 kb was gained. The activity of ALP was increased obviously by the AdCMV-BMP-7-infected marrow stromal cells, and the increase showed a dose and time dependence. Whereas the marrow stromal cells infected with blank adenovirus vector had no influence on ALP, indicating the marrow stromal cells were differentiated into osteoblasts after activated by AdCMV-BMP-7. CONCLUSION： Recombinant AdCMV-BMP-7 can stimulate rabbit bone marrow stromal cells to differentiate to osteoclasts in a time- and dose-dependent manner.

关 键 词：骨形态发生蛋白质类 腺病毒 骨髓细胞 细胞分化 组织构建 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]