HGF慢病毒载体的构建及其转染脂肪来源间充质干细胞的实验研究  

Construction of Lentivirus Vector Carrying HGF and Exploration of Transfection Condition of Rat Adipocyte-derived Mesenchymal Stem Cells

作　　者：张加敏[1] 冯非儿[1] 王谦明 朱晓璐[1] 王辰骢 裴博阳 张晓辉[1] 

机构地区：[1]北京大学人民医院北京大学血液病研究所,北京100044

出　　处：《中国实验血液学杂志》2015年第2期490-494,共5页Journal of Experimental Hematology

基　　金：国家科技支撑计划(2012BAI138B03);国家自然科学基金(81270643);教育部高等学校博士学科点专项科研基金(20120001110026);北京市自然科学基金(7132194);北京市科技计划项目(Z111107058811024)

摘　　要：目的:本研究旨在构建携带人肝细胞生长因子(hepatocyte growth factor,HGF)基因的慢病毒载体,探索HG F慢病毒转染脂肪间充质干细胞(adipose derived mesenchymal stem cells,ADSC)的方法。方法:应用PCR法从带有HGF基因的质粒上钓取目的基因,克隆到GV287载体中。将重组GV287-HGF载体质粒和慢病毒包装质粒共转染293T细胞,测定病毒滴度,并转染ADSC。结果:HGF基因PCR产物电泳结果与预期大小一致,重组GV287-HGF质粒PCR产物电泳结果正确,酶切后产物测序分析所得结果与目的基因序列一致,含有HGF基因的重组慢病毒构建正确。通过ELISA法测得病毒滴度为5×108TU/ml。HG F重组慢病毒转染ADSC的最佳感染复数(multiplicity of infection,MOI)值为50。结论:本研究构建表达人HGF的慢病毒载体并成功转染ADSC,为进一步研究过表达HGF的ADSC,治疗放射引起的骨髓及重要脏器损伤奠定基础。Objective：This study was to construct the lentivirus vector carrying hepatocyte growth factor （HGF） gene and to explore the condition for transfecting the adipocyte-derived mesenchymai stem cells （ ADSC ） by HGF lentivirus. Methods：The target gene was obtained from plasmid carrying HGF gene by PCR and was cloned into GV287 vector. The recombinant GV287-HGF vector plasmid and lentivirus-packing plasmid were co-transfected into 293 T cells to generate HGF lentivirus, and the virus titer was assayed, then the ADSC were transfected by using recombinant HGF lentivirus, and the optimal multplicity of infection （MOI） was detected. Results：The PCR product of HGF gene was consistent with expectant sizes, suggesting that the electrophoretic result of recombinant GV287-HGF plasmid PCR product was correct. The sequencing analysis of cleaved product showed consistance of obtained results with the sequences of target gene, suggesting correct construction of recombinant lentivirus carrying HGF gene. The ELISA showed that the virus tilter was 5 x 108 TU/ml. The optimal MOI for transfecting ADSC with recombinant lentivirus carrying HGF gene was 50. Conclusion： The lentivirus vector expressing human HGF gene has been constructed, and transfected the ADSC succesfully. This study lays a foundation for further stadying the ADSC overexpressioning HGF, treating the radiation damage of boue marrow and impartattt interrtal organs.

关 键 词：HGF ADSC 慢病毒 骨髓损伤 

分 类 号：Q78[生物学—分子生物学]