| Purpose:The purpose of this research was to analysis the mechanism of angiogenesis enhanced by the regenerated silk fibroin film modified with transfected human corneal epithelial cells(HCECs). Constructing the recombinant adenoviruses Ad-hIL-17F, then the growth-suppressing effect of Ad-hIL-17F on the corneal neovascularization to be observed.Methods:(1)To investigate the biocompatility of regenerated silk fibroin film(SF) and corneal tissue.In vitro, human corneal epithelial cells(HCECs) were cultured on SF. Then the modality of HCECs were observed by optical microscope and scanning electron microscope. Growth curve of HCECs were measure by MTT. Expression of VEGF,Ang1,EGF,TGF-βautocrined by HCECs was measure by ELISA. In vivo, regenerated silk fibroin films were implanted in the corneal stroma, the conjunctival congestion,corneal edema and the area of neovessels were recorded .The expression of CD34, collagen-I,collagen-III in corneal stroma were detected by immunohistochemistry. (2)In vitro, HCECs gene transfection by Ad-VEGF165 were cultured on SF. The transcription and expression of VEGF in HCECs gene transfection by Ad-VEGF165 was determined by real-time quantitative PCR and Western Blot. The expression of VEGF,Ang1,EGFand TGF-βin HCECs was assessed by ELISA. In vivo, regenerated silk fibroin films modified by transgenes cells were implanted in corneal stroma of rabbit , the conjunctival congestion,Corneal edema and the area of neovessels were recorded . The expression of CD34, collagen-I,collagen-III,VEGF,TGF-βand MMP-2 in corneal stroma was also examined by immunohistochemistry. (3) Inhibition of corneal neovascularization by Ad-hIL-17F . Ad-hIL-17F was assaied by RT-PCR and indirect immunofluorescence methods. The growth-suppressing effect of Ad-hIL-17F on the ECV304 cells was assessed by MTT. The transcription and expression of VEGF in ECV304 cells effect of Ad-hIL-17F was determined by ELISA, real-time quantitative PCR. Regenerated silk fibroin films modified by transgenes cells were implanted in corneal stroma. The in vivo enhanced growth-suppressing effect of Ad-hIL-17F on corneal neovessels by subconjunctival injection at one week after operation. The conjunctival congestion and the area of neovessels were recorded. Expression of CD34 and VEGF in corneal stroma were detected by immunohistochemistry.RESULTS:(1) corneal epithelium cells grow well in regenerated silk protein thin film. Rabbit corneal stroma implanted with regenerated silk protein thin film remain transparent with no obvious neovascularization nor implant falling off, no infection and no inflammatory reactions in the anterior chamber of the eye. (2)In the culture plate of Ad-VEGF165 transgenic corneal epithelium cells, the expression of VEGF increases, meanwhile, the expression of neovascularization related growth factors such as Ang1,EGF,TGF-βetc. also increased. After the implantation of regenerated silk protein film loaded with Ad-VEGF165 transgenic corneal epithelium cells, cornea healing reaction is strong, inflammatory cells infiltrates, collagen hyperplasia, the expression of neovascularization related cell factors such as VEGF,MMP-2,TGF-βincrease. The implantion of regenerated silk protein film loaded with Ad-VEGF165 transgenic corneal epithelium cells can induce neovascularization in cornea and the new vessels can sustain more than one month. (3) Sequencing results indicate that the sequence of hIL-17F is correct. The expression of IL-17F in QBI-293A cells are detected by RT-PCR and indirect immuno-fluorescence methods. Ad-hIL-17F suppresses the growth of ECV304 cells. It suppresses the transcription of VEGF and the expression of the gene of VEGF,Ang-1 in ECV304 cell.. Injection of Ad-hIL-17F in subconjunctiva of a rabbit's eye can effectively supress neovascularization in cornea induced by Ad-VEGF165 transgenic corneal epithelium cells regenerated silk protein film, without adverse reactions such as corneal perforation ,endophthalmitis.CONCLUSIONS:(1) The biocompatibility of regenerated silk protein film and corneal tissue is good. Regenerated silk protein film does not up-regulate neovascularization related growth factors. Implantation of regenerated silk protein film in rabbit eyes does not induce corneal vascularization because of the good biocompatibility of the implanted material with corneal tissue and thus does not initiate the process of neovasularization.(2) After the implantion of regenerated silk protein film loaded with Ad-VEGF165 transgenic corneal epithelium cells into rabbit's eye, expression of VEGF,MMP-2,TGF-β,CD34 etc. in corneal stroma all increase. Regenerated silk protein film has good cell adhesion property, thus can immobilize cells on the material surface. Induce continuous expression of angiogenesis related growth factors via transgenic cells, thus introduces concentration gradient in local tissues, and eventually induced tissue neovasularization.(3) Ad-hIL-17F significantly down-regulates the expression of VFGF in ECV304 cells. Injection of Ad-hIL-17F in subconjunctiva suppresses corneal vascularization, this may be related with the suppression of the transcription and expression in vascular endothelial cell.
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