| 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 |
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| 引用本文： | 周明,石新艳,田少奇,王丽,张积华,孙康,邢士超,孙伟雪,黄洪杰,吴丹. 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[J]. 中国修复重建外科杂志, 2011, 0(4): 482-487 |
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| 作者姓名： | 周明 石新艳 田少奇 王丽 张积华 孙康 邢士超 孙伟雪 黄洪杰 吴丹 |
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| 作者单位： | 青岛大学医学院附属医院关节外科;青岛大学医学院附属医院医用材料办公室;青岛大学医学院附属医院中美干细胞与再生医学中心;青岛大学医学院附属医院查体中心;青岛大学医学院附属医院中心实验室;青岛大学医学院附属医院整形美容烧伤科; |
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| 基金项目： | 山东省科技攻关计划资助项目(2008GG30002037)~~ |
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| 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。
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| 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 |
CONSTRUCTION OF INDUCIBLE LENTIVIRAL VECTOR CONTAINING HUMAN BONE MORPHOGENETIC PROTEIN 2 GENE AND ITS EXPRESSION IN HUMAN UMBILICAL CORD BLOOD MESENCHYMAL STEM CELLS |
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| ZHOU Ming,SHI Xinyan,TIAN Shaoqi,WANG Li,ZHANG Jihua,SUN Kang,XING Shichao,SUN Weixue,HUANG Hongjie,WU Dan.Joint Surgery,Medical Materials Office,Chinese-American Research Center for Stem Cells , Regenerative Medicine,Health Physical Examination Center,Central Laboratory,Plastic Surgery,the Affiliated Hospital of Medical College,Qingdao University,Qingdao Sh,ong,,P.R.China.. CONSTRUCTION OF INDUCIBLE LENTIVIRAL VECTOR CONTAINING HUMAN BONE MORPHOGENETIC PROTEIN 2 GENE AND ITS EXPRESSION IN HUMAN UMBILICAL CORD BLOOD MESENCHYMAL STEM CELLS[J]. Chinese journal of reparative and reconstructive surgery, 2011, 0(4): 482-487 |
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| Authors: | ZHOU Ming SHI Xinyan TIAN Shaoqi WANG Li ZHANG Jihua SUN Kang XING Shichao SUN Weixue HUANG Hongjie WU Dan.Joint Surgery Medical Materials Office Chinese-American Research Center for Stem Cells Regenerative Medicine Health Physical Examination Center Central Laboratory Plastic Surgery the Affiliated Hospital of Medical College Qingdao University Qingdao Sh ong P.R.China. |
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| Affiliation: | ZHOU Ming1,SHI Xinyan2,TIAN Shaoqi1,WANG Li3,ZHANG Jihua4,SUN Kang1,XING Shichao5,SUN Weixue1,HUANG Hongjie1,WU Dan6.1Joint Surgery,2Medical Materials Office,3Chinese-American Research Center for Stem Cells and Regenerative Medicine,4Health Physical Examination Center,5Central Laboratory,6Plastic Surgery,the Affiliated Hospital of Medical College,Qingdao University,Qingdao Shandong,266003,P.R.China. |
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| Abstract: | 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 cell... |
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| Keywords: | Inducible lentiviral vector Human bone morphogenetic protein 2 Tet-on system Human umbilical cord blood mesenchymal stem cells Doxycline |
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