Research On Effect Of Regenerated Silk Fibroin Film On Transcription And Expression Of VEGF Gene

Objective: On the observation on the biological behaviour of ECV304and the L929-VEGF transgenic cell cultivated on regenerated silk fibroinfilm, the effect of regenerated silk fibroin film on transcription andexpression of vascular endothelial growth factor gene is studied.Methods: Regenerated silk fibroin film was tested by toxic experimentand adhesion experiment, the influence on cell cycle and apoptosis of thefibroblast that culviated on regenerated silk fibroin film was detected byflow cytometry ( FCM ). ECV's growth shape and growth curve onregenerated silk fibroin film were observed, and the influence on VEGFsecretion of vascular endothelial cell that culviated on regenerated silkfibroin film was detected by ELISA test. The cDNA fragment encodinghVEGF165 was amplified by RT-PCR.Then the recombinant eukaryoticexpression plasmid was constructed and identified by endonucleasedigestion. The cDNA fragment was sequenced. The E929 was infected bypcDNA3.0-VEGF respectively, and the positive L929 was obtained byselection with G418. Then the L929-VEGF transgenic cell's growth shapeon regenerated silk fibroin film was observed,real-time quantitative reversetranscriptase polymerase chain reaction (RQ-RT-PCR) method and ELISAtest were used to detect the transcription and expression of VEGF gene.Results: Regenerated silk fibroin film has not cytotoxicity, and thefibroblast adheres well on regenerated silk fibroin film. The film doesn'thave adverse influence on cell cycle and apoptosis of the fibroblast that culviated on the film.And it doesn't interfere the growth and the VEGFsecretion of vascular endothelial cell. The full encoding sequence ofhVEGF165 was obtained and identified with that included in GenBank.Expression vectors were constructed correctly. The transgenic cell growswell on regenerated silk fibroin film, and RQ-RT-PCR and ELISA testconfirm that regenerated silk fibroin film doesn't interfere transgenic cell'sVEGF gene's transcription and expression.Conclusion:Regenerated silk fibroin film doesn't have adverseinfluence on the growth of the fibroblast and the vascular endothelial cellthat culviated on the film.And it does not interfere the transcription andexpression of vascular endothelial growth factor gene. It shows thatregenerated silk fibroin film is an excellent biomaterial with goodbiocompatibility.