Study On Inducing Angiogenesis Of Regenerated Silk Fibroin Film Modified By The Ang-1 Or/and VEGF165 Transgenic Cells And Its Mechanism

Objective:To construct expressing angiogenin-1(Ang-1) or/and vascular endothelial growth factor (VEGF165) recombinant adenoviral vectors and explore the effect of the regenerated silk fibroin film (SF) modified by their transgenic fibroblasts on inducing angiogenesis and its mechanism.Methods:The VEGF165 fragments were amplified by PCR using pcDNA3.0-VEGF165 plasmids as templates and the Ang-1 fragments were amplified by RT-PCR using the total RNA from human bone marrow cells as templates. Then the VEGF165 (XhoI,EcoRⅤ) and Ang-1 (BglII,SalI) fragments were subcloned into pAdTrack-CMV-IRES transfer vector and identified by PCR, double endonuclease digestion, and DNA sequencing. The pAdTrack-CMV-IRES-VEGF165, pAdTrack-CMV-Ang-1-IRES and pAdTrack-CMV-Ang-1-IRES-VEGF165 transfer vectors linearized with PmeI digestion and pAdEasy-1 backbone vector were further cotransformed into the bacteria BJ5183 competent cells for homologous recombination. The homologous recombinant plasmids were transformed into the bacteria DH5αcells competent cells to abundantly amplify the plasmids. Then they were linearized with PacI digestion and transfected into the human embryonic kidney 293 (293A) cells by Lipofectamine2000, leading to formation of the recombinant adenoviruses Ad-VEGF165, Ad-Ang-1, and Ad-Ang-1-VEGF165. The morphology of 293A cells infected with Ad-VEGF165, Ad-Ang-1, and Ad-Ang-1-VEGF165 on the SF, polyvinyl chloride film (PVC), polyethylene film (PE) different materials was observed under an inverted microscope.The transgene expression was examined by enzyme-labeled immunosorbent assay (ELISA). Meanwhile, the VEGF, Ang-1, fibroblast growth factor 2 (FGF2), and platelet-derived growth factor (PDGF) expression levels of WI-38 cells infected with Ang-1 or/and VEGF165 on SF were detected by ELISA and the gene expression profile of WI-38 cells infected with Ad-Ang-1-VEGF165 on SF was assessed by gene chip. The effect of Ang-1 or/and VEGF165 on the angiogenesis of the chick chorioallantoic membrane was assessed by chorioallantoic membrane (CAM) assay. Wounds with full-thickness cutaneous defect were produced on the dorsum of the SD rats.The rats were randomly divided into four groups: SF group, SF+MSC (SFMSC) group, SF+MSC+Ad-Ang-1-VEGF (SFMSCAV) group and PBS group.In the experimental group,the wounds were covered with SF membrane planted with MSC modified by Ad-Ang-1-VEGF.For the three control groups,the wounds were covered with SF membrane planted by the MSC,blank SF membrane, and no membrane,respectively.The process for granulation tissue formation and wound healing were general observed.Specimens were obtained at 7 and 14 days after injury,and immunohistochemistry was employed to observe the angiogenesis and the process for granulation tissue formation.Results:The VEGF165 and Ang-1 fragments amplified by PCR were subcloned into pAdTrack-CMV-IRES transfer vector and the target genes identified by DNA sequencing were the same as the GenBank reports. The adenoviral vectors expressing Ang-1 or/and VEGF165, Ad-VEGF165, Ad-Ang-1, and Ad-Ang-1-VEGF165 vectors were successfully constructed and obtained. The aim gene can be expressed in 293A cells on SF. Meanwhile, not only the target genes Ang-1 or/and VEGF165 which accelerates angiogenesis expression levels of WI-38 fibroblasts infected with Ang-1 or/and VEGF165 cultured on the SF were enhanced (P<0.05), but also the FGF2 and PDGF which are related to wound healing could stably express in the transgenic WI-38 cells on the SF as the control group. The gene chip result showed that SF was not only advantageous to the expression of cell adhesion genes and the growth factor-related genes, but also enhanced the angiogenesis and tissue-repair-related genes expression of the Ad-Ang-1-VEGF165-transgenic WI-38 cells. CAM assay suggested that the SF modified by Ang-1 or/and VEGF165 transgenic fibroblasts had a marked proangiogenesic activity. The wound healing experiment further proved that the SF modified by Ad-Ang-1-VEGF165 transgenic MSC was beneficial for angiogenesis and the reconstruction of damaged tissues. Conclusion:The adenoviral vectors expressing Ang-1 or/and VEGF165 were successfully constructed. We demonstrated that adenoviral vectors not only can be efficiently infected into fibroblasts cultured on the SF, but also the FGF2 and PDGF could stably express in the transgenic fibroblasts on the SF. The SF modified by Ang-1 or/and VEGF transgenic fibroblasts had a marked proangiogenesic activity. It provided experimental basis for modifying SF of inducing angiogenesis. Moreover, it also lays a foundation for the future research on preparing the biomedical materials for inducing angiogenesis by adopting genetic technology.