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目的研究经腺病毒介导的人血管内皮生长因子165(VEGF165)转基因细胞修饰的再生丝素膜(SF)诱导血管形成活性及其分子机制。方法将WI-38人胚肺成纤维细胞分别培养在有(SFC组)或无(PBS组)再生丝素膜的24孔培养板上,24h后用带绿色荧光蛋白(GFP)基因的空载体腺病毒Ad-GFP(Ad-GFP组/SFCG组)或含有VEGF165目的基因的重组腺病毒Ad-VEGF165(Ad-VEGF165组/SFCV组)感染培养细胞。通过形态学观察和ELISA法检测各组细胞培养上清液中VEGF165目的基因表达及其对细胞自分泌的血管生成素-1(Ang-1)、碱性成纤维细胞生长因子(FGF2)、血小板衍化生长因子(PDGF)表达的影响,并用鸡胚绒毛尿囊膜(CAM)和在兔眼角膜上进行新生血管诱导实验。结果形态学观察发现WI-38细胞在再生丝素膜上不仅贴壁生长良好,而且易被Ad-GFP、Ad-VEGF165腺病毒感染,呈现强荧光。再生丝素膜利于Ad-VEGF165组中VEGF165目的基因在细胞中的高表达(P<0.05),而且还可使细胞自分泌的Ang-1表达水平明显升高(P<0.05),同时可维持细胞自分泌的FGF2及PDGF稳定表达。Ad-VEGF165组(SFCV组)具有促进CAM上血管生长活性和诱导兔眼角膜组织新生血管生成作用。结论 Ad-VEGF165感染WI-38成纤维细胞所修饰的再生丝素膜,具有诱导血管新生的功能。
Objective To investigate the angiogenic activity and molecular mechanism of adenovirus-mediated regeneration of silk fibroin (SF) -mediated vascular endothelial growth factor 165 (VEGF165) transgenic cells. Methods WI-38 human embryonic lung fibroblasts were cultured in 24-well plates with (SFC group) or without (PBS group) regenerated silk fibroin membrane respectively. After 24 hours, empty vector with green fluorescent protein (GFP) gene Cultured cells were infected with adenovirus Ad-GFP (Ad-GFP / SFCG) or recombinant adenovirus Ad-VEGF165 (Ad-VEGF165 / SFCV) containing VEGF165 gene. Morphological observation and ELISA were used to detect the expression of VEGF165 gene in the cell culture supernatant of each group and its effect on the autocrine angiopoietin-1 (Ang-1), basic fibroblast growth factor (FGF2) (PDGF) expression. Chick embryo chorioallantoic membrane (CAM) and neovascularization experiments were performed on rabbit cornea. Results Morphological observation showed that WI-38 cells not only adherently grew well on the regenerated silk fibroin membrane, but also were easily infected with Ad-GFP and Ad-VEGF165 adenovirus and showed strong fluorescence. The regenerated silk fibroin membrane was beneficial to the high expression of VEGF165 gene in Ad-VEGF165 group (P <0.05), but also the autocrine Ang-1 expression was significantly increased (P <0.05) Cell autocrine FGF2 and PDGF are stably expressed. Ad-VEGF165 group (SFCV group) can promote angiogenic activity on CAM and induce corneal neovascularization in rabbit eyes. Conclusion Ad-VEGF165 infects WI-38 fibroblasts modified silk fibroin membrane and induces angiogenesis.