再生丝素膜对Ad-VEGF_(165)转基因成纤维细胞基因表达的影响(英文)

Effects of regenerated silk fibroin film on cytokine expression in the Ad-VEGF_(165) transgenic fibroblasts

背景:前期实验已证实再生丝素膜可促进pcDNA3.0-VEGF165转染的L929细胞表达血管内皮生长因子(Vascular endothelial growth factor,VEGF)。目的:进一步观察再生丝素膜对经腺病毒介导的人VEGF(Ad-VEGF165)转基因成纤维细胞基因转录表达的影响。设计、时间及地点:对比观察细胞基因工程实验,于2007-11/2008-04在苏州大学细胞与分子生物实验室完成。材料:再生丝素膜由苏州大学材料工程学院李明忠教授提供。方法:将Ad-VEGF165腺病毒感染培养在再生丝素膜、聚氯乙烯膜及聚乙烯膜上的QBI-293A人胚肾和WI-38人胚肺成纤维细胞,以空载体Ad-GFP腺病毒和未接种病毒的PBS组为对照。主要观察指标:通过实时定量RT-PCR法检测不同材料对成纤维细胞VEGF mRNA转录的影响;ELISA法检测其对VEGF、血管生成素1、碱性成纤维细胞生长因子、血小板衍化生长因子表达的影响。结果:再生丝素膜组成纤维细胞VEGF mRNA呈高水平表达,但聚氯乙烯膜组转录水平明显下降(P<0.05)。再生丝素膜组Ad-VEGF165转基因293A细胞及WI-38细胞不仅目的基因VEGF细胞因子的分泌呈高水平表达,并可促使血管生成素1基因表达水平明显提高(P<0.05),而且再生丝素膜还可使成纤维细胞自身分泌的碱性成纤维细胞生长因子、血小板衍化生长因子呈现正常表达水平。结论:再生丝素膜不仅利于VEGF目的基因在成纤维细胞中呈现高效表达,并促进血管生成素1基因的表达,而且能维持成纤维细胞自身分泌的与组织损伤修复相关基因碱性成纤维细胞生长因子、血小板衍化生长因子的正常表达。

BACKGROUND： Previous studies have demonstrated that regenerated silk fibroin film can promote pcDNA3.0-vascular endothelial growth factor 165 （VEGF165） transfected L929 cells to express VEGF. OBJECTIVE： To investigate the effects of regenerated silk fibroin film on expression of cytokines related to angiogenesis in the fibroblasts transfected by adenovirus mediated-VEGF165 （Ad-VEGF165）. DESIGN, TIME AND SETTING： Controlled observational cell gene engineering experiment performed by analysis of variance at the Laboratory of Cellular and Molecular Biology, Soochow University between November 2007 and April 2008. MATERIALS： Regenerated silk fibroin film was provided by Professor Li Ming-zhong, who was from Department of Material Science and Engineering, Soochow University. METHODS： The QBI-293A and WI-38 fibroblasts cultured on the regenerated silk fibroin film, polyvinyl chloride film and polyethylene film were transfected by Ad-VEGF165. Cells were also transfected by adenovirus-green fluorescent protein （Ad-GFP） and treated by phosphate buffered saline （PBS） for controls. MAIN OUTCOME MEASURES： VEGF mRNA was detected by real-time reverse transcription-polymerase chain reaction （RT-PCR）; the expression levels of VEGF, angiogenin 1（Ang 1）, fibroblast growth factor 2 （FGF2）, and platelet-derived growth factor （PDGF） were detected by enzyme-labeled immunosorbent assay （ELISA）. RESULTS： The VEGF mRNA expression in the fibroblasts cultured on the regenerated silk fibroin film was increased but that in the fibroblasts cultured on the polyvinyl chloride film was significantly decreased （P 〈 0.05）. ELISA results demonstrated that not only VEGF gene expression in 293A and WI-38 cells transfected by Ad-VEGF165 cultured on regenerated silk fibroin film was high, but also Ang 1 expression increased significantly （P 〈 0.05）. Meanwhile, the expression levels of FGF2 and PDGF were normal in the fibroblasts cultured on the regenerated silk fibroin film. CONCLUSION： Adenovirus vector can be efficiently transfected into fibroblasts cultured on the regenerated silk fibroin film and can express VEGF and Ang 1 protein with highly biological activity, which accelerates angiogenesis. Regenerated silk fibroin film also can maintain the normal expression levels of FGF2 and PDGF, which are related to wound healing.