摘要
目的:构建含人肝细胞生长因子(hHGF)基因的骨髓间充质干细胞(MSCs),获得高表达hHGF的MSCs。方法:构建含hHGF基因的腺病毒表达载体pDC316-HGF-IRES-EGFP。同源重组法获得含hHGF的重组腺病毒Ad-HGF。以仅表达GFP基因的重组腺病毒Ad-GFP为对照,体外分别以Ad-GFP,Ad-HGF感染MSCs。荧光激活细胞分选术检测受染细胞的基因表达效率。酶联免疫吸附测定法检测转导细胞培养上清中hHGF表达的浓度和持续时间。结果:腺病毒表达载体经限制性内切酶切后有2.2 kb的hHGF目的基因片段和5.2 kb的线性化载体片段;目的片段测序结果与基因库中hHGFcDNA序列一致。病毒液Ad-HGF纯化后滴度可以达到8.02×1010pfu/ml。FACS检测HGF/MSCs的GFP阳性率达95.19%。HGF/MSCs细胞培养上清中hHGF能持续表达至少14 d;最高浓度达到99 ng/ml。结论:本实验中腺病毒表达载体构建正确;重组腺病毒液Ad-HGF滴度高;转导后在MSCs中获得高效、稳定和持续表达的hHGF。该载体达到实验设计要求,为进一步的体内外研究提供了实验依据。
Objective： To construct the adenoviral expression vector system containing human HGF cDNA, and produce the virus containing hHGF, and to further study the transducing efficiency and the expression of HGF in MSCs. Methods： The HGF cDNA was amplificated from the expression plasmid pCMV- HGF by PCR,and subcloned into the same site of the plasmid pDC316-IRES-EGFP vector. The recombinants, named pDC316-HGF-IRES-EGFP, were identificated with restriction enzyme digestion and sequencing. Then we produced virus Ad-HGF and Ad-GFP by homologous recombination in HEK293 package cells. BM-MSCs were transduced with Ad-HGF, and HGF/MSCs were generated. The efficiency of Ad- HGF transduction was assessed by FACS analysis. The HGF concentrations in supernatants of HGF/MSCs were determined by ELISA. Results： The recombinants pDC316-HGF-IRES-EGFP contained fragments of hHGF. The DNA sequencing of HGF was identical to the report in Genbank. After packing of the pDC316- HGF-IRES-EGFP in HEK293 ceils, 8.02 × 10^10 pfu/ml titer of Ad-HGF was obtained. Forty-eight hours after transduction, 95.19% of HGF/MSCs were GFP positive, and peak concentration levels of hHGF in the cultured supernatants were detected. The adenovirus-mediated expression of HGF by MSCs was maintained for at least 2 weeks in vivo. Conclusion： Our data demonstrated that the adenoviral expression vector system pDC316-HGF-IRES-EGFP containing hHGF cDNA had been constructed correctly, and its effective expression also had been obtained in MSCs. It will provide material basis for the next study.
出处
《江苏大学学报（医学版）》
CAS
2009年第4期324-327,341,I0002,共6页
Journal of Jiangsu University：Medicine Edition
关键词
间充质干细胞
肝细胞生长因子
腺病毒载体
mesenchymal stem cells
hepatocyte growth factor
adenoviral expression vector
