核心结合因子α1过表达慢病毒载体的构建及其在人牙周膜成纤维细胞中的表达和意义  

Reconstruction of recombinant lentiviral vector containing human CBFα1 gene and its expression in human periodontal ligament fibroblasts

作　　者：曹金芳[1] 邓辉[1] 金向青[1] 

机构地区：[1]温州医学院附属口腔医院口腔内科,325027

出　　处：《浙江医学》2012年第2期87-90,共4页Zhejiang Medical Journal

基　　金：浙江省教育厅资助项目（20070914）;温州市科技局资助项目（Y20070052）

摘　　要：目的 构建含人核心结合因子α1（CBFα1）基因的过表达慢病毒载体,并观察在重组慢病毒感染的人牙周膜成纤维细胞（PDLFs）中CBFα1基因的表达情况.方法 采用PCR 技术体外扩增人CBFα1,将扩增产物与慢病毒载体pGC-FU连接,构建重组质粒pGC-FU-hCBFα1,并进行酶切及测序鉴定.利用脂质体转染法将pGC-FU-hCBFα1、pHelper1.0和pHelper2.0的3种质粒共转染293T细胞,包装产生慢病毒.将PDLFs分为未感染组（未感染任何病毒）、阴性对照病毒感染组（加阴性对照病毒感染）、CBFα1重组慢病毒感染组（加CBFα1重组慢病毒感染）,应用免疫细胞化学方法检测目的 基因CBFα1在PDLFs中的表达.结果 重组质粒经测序证实,插入片段与人CBFα1基因序列完全一致.包装慢病毒后检测病毒滴度为2.00×109 TU/ml.免疫细胞化学检测证实了CBFα1在PDLFs中的有效表达.结论 成功构建了携带hCBFα1基因的重组慢病毒,并能有效感染PDLFs,为进一步研究其在牙周组织再生中的生物学功能奠定了基础.Objective To construct a recombinant lentiviral vector containing human CBFa 1 gene and to investigate its expression in human periodontal ligament fibroblasts. Methods A lentiviral expression vector for human CBFa 1 was constructed by recombinant DNA technique. The reconstructed lentiviral vector containing hCBF a 11 was confirmed by PCR and sequencing, The human periodontal ligament fibroblast （PDLF） 293T cells were cotransfected with lentiviral vector pGC-FU-hCBFa 1, pHelper 1.0 and pHelper2.0 with Lipofectamine 2000. PDLFs were infected with recombinant lentiviral vector containing CBF a 1. Infection efficiency was measured by fluorescent microscope and CBFa 1 expression was detected by immunocytochemical analysis. Results The sequence analysis of recombinant plasmid pGC-FU-hCBFa 1 showed that the human CBFa 1 was inserted into lentiviral vector pGC-FU accurately. The titer of concentrated virus was 2.00 × 109TU/mI. Immunohistochemistry showed that CBFa 1 was expressed in PDLF 293T cells infected with recombinant lentiviral vector for CBF a 1. Conclusion The recombinant lentiviral vector pGC-FU-hCBF a 1 has been constructed and CBF a 1 are expressed in transfectecl PDLFs successfully.

关 键 词：核心结合因子Α1 慢病毒载体 转染 感染 

分 类 号：R781.4[医药卫生—口腔医学]