Ad-Ang-1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

Study on Inducing Angiogenesis of Regenerated Silk Fibroin Film Modified by the Ad-Ang-1 Transgenic Fibroblasts

目的研究经腺病毒介导的人血管生成素-1（Ad-Ang-1）转基因细胞修饰的再生丝素膜诱导血管形成活性及其分子机制。方法将Ad-Ang-1腺病毒病毒子感染再生丝素膜上培养的wI-38人胚肺成纤维细胞，通过ELISA法检测再生丝素膜对成纤维细胞目的基因表达的影响，将Ad—Ang-1转基因WI-38细胞修饰的丝素材料进行鸡胚尿囊膜血管形成实验（CAM）和兔角膜层间植入诱导角膜新生血管试验，并用ELISA法检测其对血管内皮生长因子（VEGF）、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。结果Ad—Ang-1可感染再生丝素膜上培养的成纤维细胞，ELISA法检测结果表明sF组Ad．Ang．1转基因W1．38细胞可以成功表达目的基因（P〈0．05）。CAM实验表明Ad-Ang-l转基因wI-38细胞修饰的再生丝素膜具有促进CAM血管生成的活性，并通过兔角膜层间植入实验进一步证明Ad-Ang-1转基因细胞修饰的丝素材料具有诱导角膜组织血管生成的作用．其机制可能与再生丝素膜对Ad-Ang-l转基因的WI-38成纤维细胞中不仅可以成功表达目的Ang-1，而且还可使与血管生成和组织损伤修复相关的VEGF、FGF2、PDGF基因表达水平明显提高有关。结论Ad-Ang-I转基因细胞修饰的丝素材料具有诱导血管生成的作用，为今后进一步采用转基因技术对生物材料进行修饰或改性、研制出具诱导性的医用生物膜材料创造了条件。

Objective To study the regenerated silk fibroin film（SF） as a biomedical material,we investigate its effects modified by the fibroblasts infected by adenovirus mediated-Ang-l（Ad-Ang-l） on inducing angiogenesis. Methods The WI-38 fibroblasts cultured on the SF and no film infected with Ad-Ang-l,they were infected with adenovirus-green fluorescent protein（Ad-GFP） and treated by phosphate buffered saline（PBS）.We observed the cell growth morphology and detected the expression of aim gene Ang-l.The effect ofAd-Ang-1 on the angiogenesis of the chick chorioallantoic membrane was assessed by chorioallantoic membrane（CAM） assay and that inducing corneal neovascularization through the SF modified by transgenic cells implanted into thecorneal stroma of rabbits.The expression levels of vascular endothelial growth factor（VEGF）,fibroblast growth factor 2（FGF2）,and platelet-derived growth factor（PDGF） were detected by enzyme-labeled immunosorbent assay（ELISA）.Results Adenovirus vector can be efficiently infected into fibroblasts cultured on the SF; the expression levels of Ang-1 in WI-38 cells infected by Ad-Ang-1 cultured on the SF enhanced（P〈0.05）;CAM assay suggested that the SF modified by Ad-Ang-1 transgenic fibroblasts had a marked promoting angiogenesis activity.The SF modified by transgenic cells implanted into the corneal stroma of rabbits can induce corneal neovascularization.The ELISA results showed that the expression of VEGF, FGF2, and PDGF increased significantly in the Ad-Ang-1 transgenic WI-38 fibroblasts（P〈0.05）. Conclusion The pbssible reasons why the SF modified by transgenic fibroblasts can induce angiogenesis may be that it can express VEGF and Ang-1 with highly biological activity and increase the expression of FGF2 and PDGF which are related to angiogenesis.It lays a foundation for future research on the biomedical materials of inducing angiogenesis.