Zhang Jia-Ming, Feng Fei-Er, Wang Qian-Ming, Zhu Xiao-Lu, Wang Chen-Cong, Pei Bo-Yang, Zhang Xiao-Hui
Peking University Institate of Hematology, Peking University People Hospital, Beijing 100044, China.
Peking University Institate of Hematology, Peking University People Hospital, Beijing 100044, China. E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2015 Apr;23(2):490-4. doi: 10.7534/j.issn.1009-2137.2015.02.038.
This study was to construct the lentivirus vector carrying hepatocyte growth factor (HGF) gene and to explore the condition for transfecting the adipocyte-derived mesenchymal stem cells (ADSC) by HGF lentivirus.
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.
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 5×10(8) TU/ml. The optimal MOI for transfecting ADSC with recombinant lentivirus carrying HGF gene was 50.
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 over-expressioning HGF, treating the radiation damage of bone marrow and impartant internal organs.
构建携带肝细胞生长因子（HGF）基因的慢病毒载体，并探索HGF慢病毒转染脂肪来源间充质干细胞（ADSC）的条件。
通过PCR从携带HGF基因的质粒中获取目的基因，并克隆至GV287载体。将重组GV287-HGF载体质粒与慢病毒包装质粒共转染至293T细胞以产生HGF慢病毒，测定病毒滴度，然后用重组HGF慢病毒转染ADSC，并检测最佳感染复数（MOI）。
HGF基因的PCR产物与预期大小一致，表明重组GV287-HGF质粒PCR产物的电泳结果正确。酶切产物的测序分析显示所得结果与目的基因序列一致，表明携带HGF基因的重组慢病毒构建正确。ELISA显示病毒滴度为5×10(8) TU/ml。用携带HGF基因的重组慢病毒转染ADSC的最佳MOI为50。
已构建表达人HGF基因的慢病毒载体，并成功转染ADSC。本研究为进一步研究ADSC过表达HGF、治疗骨髓及重要内脏器官的辐射损伤奠定了基础。
