Ad-VEGF165转基因成纤维细胞修饰的再生丝素膜诱导血管形成效应及其分子机制研究

Angiogenesis inducing effect and molecular mechanism of regenerated silk fibroin films modified by Ad\|VEGF165 transgenic fibroblasts

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 study the activity of angiogenesis induced by regenerated silk fibroin（SF） films modified by adenovirus／｜mediated vascular endothelial growth factor 165（Ad／｜VEGF165） and molecular mechanism.Methods WI／｜38 human embryo lung fibroblasts were cultured respectively in 24／｜hole culture plates with regenerated SF films（SFC group） or without regenerated SF films（PBS group）.After 24 hours,the cultured fibroblasts were infected by null vector adenovirus bearing green fluorescent protein gene（Ad／｜GFP,Ad／｜GFP group/SFCG group） or recombinant adenovirus containing VEGF165 target gene（Ad／｜VEGF165 group/SFCV group）.The expression of VEGF165 in supernate was observed through morphological observation.The influences of VEGF165 on the expressions of autocrine angiogenin／｜1（Ang／｜1）,basic fibroblast growth factor 2（bFGF2） and platelet／｜derived growth factor（PDGF） were detected by using ELISA in all groups.The induction test of neovascularization was carried out in chicken chorioallantioc membrane（CAM） and rabbit corneas.Results The morphological observation showed that WI／｜38 fibroblasts had a good adhesion growth in regenerated SF films,and were vulnerable to Ad／｜GFP and Ad／｜VEGF165 demonstrating hyperfluroescence.The regenerated SF films increased the expressions of VEGF165 target gene（P0.05） and autocrine Ang／｜1（P0.05）,and at the same time,kept the stable expressions of FGF2 and PDGF in Ad／｜VEGF165 group.The activity of angiogenesis in CAM was improved and neovascularization in rabbit corneas was induced in Ad／｜VEGF165 group（SFCV group）.Conclusion The regenerated SF films modified by Ad／｜VEGF165／｜infecting WI／｜38 fibroblasts can induce angiogenesis.