可诱导表达hBMP-2的慢病毒载体构建及其在人脐血间充质干细胞中的表达  

CONSTRUCTION OF INDUCIBLE LENTIVIRAL VECTOR CONTAINING HUMAN BONE MORPHOGENETIC PROTEIN 2 GENE AND ITS EXPRESSION IN HUMAN UMBILICAL CORD BLOOD MESENCHYMAL STEM CELLS

作　　者：周明[1] 石新艳[2] 田少奇[1] 王丽[3] 张积华[4] 孙康[1] 邢士超[5] 孙伟雪[1] 黄洪杰[1] 吴丹[6] 

机构地区：[1]青岛大学医学院附属医院关节外科,山东青岛266003 [2]青岛大学医学院附属医院医用材料办公室,山东青岛266003 [3]青岛大学医学院附属医院中美干细胞与再生医学中心,山东青岛266003 [4]青岛大学医学院附属医院查体中心,山东青岛266003 [5]青岛大学医学院附属医院中心实验室,山东青岛266003 [6]青岛大学医学院附属医院整形美容烧伤科,山东青岛266003

出　　处：《中国修复重建外科杂志》2011年第4期482-487,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：山东省科技攻关计划资助项目(2008GG30002037)~~

摘　　要：目的构建可诱导表达hBMP-2的慢病毒载体,并研究hBMP-2在人脐血间充质干细胞(human umbilical cord blood mesenchymal stem cells,HUMSCs)中的可诱导性表达。方法以pcDNA-hBMP-2为模板,通过PCR反应获取hBMP-2,运用GATEWAY技术构建pLV/EXPN2-Neo-TRE-hBMP-2、pLV/EXPN2-Puro-EF1A-反向反式激活因子(reverse transactivator,rtTA),通过PCR鉴定重组慢病毒载体构建。将重组慢病毒与辅助质粒共同转染293FT细胞,包装成能表达hBMP-2的可控性慢病毒,并测定病毒滴度。用病毒转染HUMSCs,使用强力霉素进行诱导,分别在相同诱导时间(48h)不同诱导浓度(0、10、100ng/mL,1、10、100μg/mL)及不同诱导时间(12、24、48、72h)相同诱导浓度(10μg/mL)两种情况下用ELISA法测定hBMP-2的表达情况。对转染前后的HUMSCs进行成骨诱导,用茜素红染色法观察矿化物结节形成情况。结果成功构建了携带hBMP-2的可控性慢病毒载体pLV/EXPN2-Neo-TRE-hBMP-2、pLV/EXPN2-Puro-EF1A-rtTA,并获得相应的病毒,病毒滴度分别为3.5×108TU/mL和9.5×107TU/mL;病毒转染HUMSCs后,HUMSCs可通过强力霉素的诱导可控性表达hBMP-2。在相同诱导时间情况下,强力霉素10μg/mL时诱导表达最强;而在相同诱导浓度下,hBMP-2的表达在诱导48h达峰值。HUMSCs成骨诱导培养2周后,茜素红染色示胞浆中有大量红色的钙化基质沉积。结论通过GATEWAY技术可以建立携带hBMP-2目的基因的可控性慢病毒载体,为进一步研究hBMP-2诱导HUMSCs成骨分化治疗骨坏死模型奠定实验基础,并提供了一种新的实验思路。Objective To construct inducible lentiviral vector containing human bone morphogenetic protein 2（hBMP-2） gene and to study its expression in human umbilical cord blood mesenchymal stem cells（HUMSCs）.Methods hBMP-2 gene was amplified by PCR from a plasmid and was cloned into pDown by BP reaction.pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-reverse transactivator（rtTA） were obtained with GATEWAY technology,and then were sequenced and analyzed by PCR.The recombinant vectors were transfected into 293FT cells respectively through lipofectamine,and the lentiviral viruses were harvested from 293FT cells,then the titer was determined.Viruses were used to infect HUMSCs in tandem.In order to research the influence of induction time and concentration,one group of HUMSCs was induced by different doxycline concentrations（0,10,100 ng/mL,and 1,10,100 μg/mL） in the same induction time（48 hours）,and the other by the same concentration（10 μg/mL） in different time points（12,24,48,and 72 hours）.The expression of target gene hBMP-2 was indentified by ELISA method.After 2-week osteogenic induction of transfected HUMSCs,the mineralization nodes were detected with Alizarin bordeaux staining method.Results The recombinant inducible lentiviral vectors（pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-rtTA） were successfully constructed.The lentiviruses were also obtained and mediated by 293FT cells,and the virus titers were 3.5 × 108 TU/mL and 9.5 × 107 TU/mL respectively.HUMSCs could expression hBMP-2 by induction of doxycycline.The expression of hBMP-2 reached the peak at 10 μg/mL doxycline at 48 hours of induction.After 2-week osteogenic induction,a lot of mineralization nodes were observed.Conclusion The recombinant inducible lentiviral vectors containing hBMP-2 gene can be successfully constructed,which provide an effective and simple method for the further study of stem cells and animal experiment in vivo.

关 键 词：可诱导慢病毒载体 HBMP-2 Tet-on系统 人脐血间充质干细胞 强力霉素 

分 类 号：R681[医药卫生—骨科学]