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

发布时间：2019-05-03 19:36

【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。
[Abstract]:Aim: to construct lentiviral vector carrying human hepatocyte growth factor (hepatocyte growth factor,HGF) gene and explore the method of HGF lentivirus transfection into adipose-derived mesenchymal stem cells (adipose derived mesenchymal stem cells,ADSC). Methods: the target gene was obtained from plasmid containing HGF gene by PCR method and cloned into GV287 vector. The recombinant GV287-HGF vector plasmid and lentivirus packaging plasmid were co-transfected into 293T cells to determine the virus titer and transfect ADSC.. Results: the results of electrophoresis of PCR products of HGF gene were consistent with the expected size. The results of electrophoresis of recombinant GV287-HGF plasmid PCR products were correct. The result of sequencing analysis after restriction endonuclease digestion was consistent with the sequence of the target gene. The recombinant lentivirus containing HGF gene was constructed correctly. The optimal complex (multiplicity of infection,MOI value of the recombinant lentivirus transfected with 5 脳 108TU/ml.HG F lentivirus was 50. The ELISA assay showed that the titer of the virus was 5 脳 (multiplicity of infection,MOI F recombinant lentivirus. Conclusion: in this study, lentiviral vector expressing human HGF was constructed and successfully transfected into ADSC, to lay a foundation for further study on the treatment of radiation-induced bone marrow and important organ injury by HGF-expressing ADSC,.
【作者单位】：北京大学人民医院北京大学血液病研究所;
【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)
【分类号】：R730.55

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