CAJ | 학술논문

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卜的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法检测其埘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甲明显下降(p<0.05).再生丝素膜组Ad-VEGF165转基因293A细胞及WI-38细胞小仅日的基因VEGF细胞因子的分泌旱高水平表达,并可促使血管生成素1基因表达水平明显提高(P<0.05),而R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:再生丝索膜不仪利于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.
배경:전기실험이증실재생사소막가촉진pcDNA3.0-VEGF165전염적L929세포표체혈관내피생장인자(Vascular endothelial growth factor,VEGF).목적:진일보관찰재생사소막대경선병독개도적인VEGF(Ad-VEGF165)전기인성섬유세포기인전록표체적영향.설계、시간급지점:대비관찰세포기인공정실험,우2007 11/2008-04재소주대학세포여분자생물실험실완성.재료:재생사소막유소주대학재료공정학원리명충교수제공.방법:장Ad-VEGF165선병독감염배양재재생사소막、취록을희막급취을희막복적QBI-293A인배신화WI-38인배폐성섬유세포,이공재체Ad-GFP선병독화미접충병독적PBS조위대조.주요관찰지표:통과실시정량RT-PCR법검측불동재료대성섬유세포VEGF mRNA전록적영향;ELISA법검측기시VEGF、혈관생성소1、감성성섬유세포생장인자、혈소판연화생장인자표체적영향.결과:재생사소막조성섬유세포VEGF mRNA정고수평표체,단취록을희막조전록수갑명현하강(p<0.05).재생사소막조Ad-VEGF165전기인293A세포급WI-38세포소부일적기인VEGF세포인자적분비한고수평표체,병가촉사혈관생성소1기인표체수평명현제고(P<0.05),이R재생사소막환가사성섬유세포자신분비적감성성섬유세포생장인자、혈소판연화생장인자정현정상표체수평.결론:재생사색막불의리우VEGF목적기인재성섬유세포중정현고효표체,병촉진혈관생성소1기인적표체,이차능유지성섬유세포자신분비적여조직손상수복상관기인감성성섬유세포생장인자、혈소판연화생장인자적정상표체.
BACKGROUND:Previous studies have demonstrated that regenerated silk fibroin film can promote pcDNA3.0-vascular endothelial growth factor 165(rEGF165) 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:ontrolled observational cell gene engineering experiment performed by analysis of variance at the Laboratory of Cellular and Molecular Biology,Soochow University between November 2007 and ApriJ 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 (Ad-GFP)and treated by phosphate buffered saline(PBS)for controls.MAlN 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 signifcantly decreased(P<0.05).ELISA results demonstrated that not only VEGF gene expression in 293A and WI-38 cells transfected bv 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 normalin the fibroblasts cultured on the regenerated silk fibroin film.CONCLUSION:Adenovirus vector can be effciently 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 ale related to wound healing.