摘要
目的构建携带大鼠肝细胞生长因子(HGF)基因的重组腺病毒载体,并转染大鼠骨髓间充质干细胞(BMSCs),检测其在BMSCs的表达、以及对BMSCs增殖和迁移的影响。方法利用全基因合成方法得到目的基因HGF序列,将HGF基因片段与穿梭质粒pDC316-mCMV-增强绿色荧光蛋白(EGFP)连接,构建含有目的基因的pDC316-mCMV-EGFP-HGF重组质粒,经过病毒包装及扩增,获得带有荧光报告基团EGFP与HGF基因的重组腺病毒载体pDC316-mCMV-EGFP-HGF。将pDC316mCMV-EGFP-HGF重组腺病毒载体转染BMSCs细胞,通过ELISA检测HGF表达水平,CCK-8法检测BMSCs增殖能力,Transwell法检测BMSCs迁移。结果构建了表达HGF基因和荧光报告基因EGFP的重组腺病毒载体pDC316-mCMV-EGFP-HGF,转染BMSCs后,BMSCs细胞HGF表达水平、增殖及迁移均明显高于非转染组(P<0.05)。结论成功构建pDC316-mCMV-EGFP-HGF重组腺病毒载体,可以介导HGF在BMSCs中稳定表达,并提高BMSCs增殖及迁移能力。
Objective To construct adenovirus vector with rat hepatocyte growth factor(HGF)gene,transfect rat bone marrow derived stem cells(BMSCs)with the adenovirus vector,and evaluate the expression of HGF in BMSCs and the impacts of adenovirus vector with HGF gene on proliferation and migration of BMSCs.Methods HGF gene fragment was connected with plasmid pDC316-mCMV-EGFP to construct the recombinant plasmid pDC316-mCMV-EGFP-HGF.After viral packaging and amplification,the adenovirus vector pDC316-mCMV-EGFP-HGF with fluorescent reporter EGFP and HGF gene was obtained.BMSCs were transfected with adenovirus vector expressing HGF gene.HGF expression was measured using ELISA,BMSCs proliferation was measured by CCK-8 assay,and BMSCs migration was assessed by Transwell method.Results The adenovirus vector pDC316-mCMV-EGFP-HGF,co-expressing HGF and EGFP gene was constructed correctly.After transfection with this adenovirus vector,HGF expression level,proliferation,and migration of BMSCs were significantly higher than those of non-transfection groups(P<0.05).Conclusion The successful construction of pDC316-mCMV-EGFP-HGF recombinant adenovirus vector can mediate the stable expression of HGF in BMSCs,and improve the proliferation and migration ability of BMSCs.
作者
罗晓红
曾雯
巨容
Luo Xiaohong;Zeng Wen;Ju Rong(Department of Neonatology,Chengdu Women's&Children's Central Hospital,School of Medicine,University of Electronic Science and Technology of China,Chengdu,Sichuan 611731,China)
出处
《四川医学》
CAS
2021年第11期1086-1090,共5页
Sichuan Medical Journal
基金
成都市科技局科技惠民项目(编号:2016-HM01-00324-SF)。
关键词
肝细胞生长因子
基因
腺病毒
骨髓间充质干细胞
增殖
迁移
hepatocyte growth factor
gene
adenovirus
bone marrow derived stem cell
proliferation
migration
