Experimental Study On VEGF Gene Transfection Restores The Angiogenesis Of Oral Submucous Fibrosis In Mice

Background:Vascular endothelial growth factor?VEGF?is closely related to the late vascular reduction of oral submucous fibrosis?OSF?,and the current application of VEGF gene transfection technology to restore angiogenesis is relatively mature.Based on the successful establishment of the mouse OSF model in previous research,this study intends to use VEGF gene transfection to restore angiogenesis of oral submucous fibrosis in mice.Objective:?1?To transfect mouse buccal fibroblasts cells in vitro by AD-EGFP-VEGF₁₆₅?adenovirus-enhanced green fluorescent protein-vascular endothelial growth factor 165?,and observe the transfection efficiency rate and the expression of human VEGF before and after transfection of each group?AD-EGFP-VEGF165 group,AD-EGFP group as experiment control,normal saline group as blank control?.?2?To explore the feasibility of VEGF gene transfection method to restore OSF angiogenesis by injecting AD-EGFP-VEGF₁₆₅ to the buccal submucosal tissue of OSF mice.Methods:?1?AD-EGFP-VEGF₁₆₅ and AD-EGFP?adenovirus-enhanced green fluorescent protein?were respectively transfected to mouse buccal fibroblasts cells cultured in vitro.The expression of human VEGF before and after transfection was detected by RT-qPCR and ELISA.The cell viability after transfection was detected by MTT.?2?Forty mice were randomly divided into experimental group?n=30?and control group?n=10?.The mice in control group were free to drink sterile water.The mice in experimental group were free to drink1000 mg/L of arecoline solution.After 20 weeks,5 mice were randomly selected from each group and their buccal tissues were excised.HE?Hematoxylin and eosin staining?,Masson,VG?Van Gieson?were used to observed the buccal tissues histological structure and determine the OSF changes in mice.?3?Fifteen OSF mice were randomly divided into 3 groups,and the same amount of AD-EGFP-VEGF₁₆₅,AD-EGFP,and normal saline were respectively injected into the buccal submucosa tissue of OSF mice.The expression of human VEGF in each group before and after transfection and the local tissue angiogenesis were observed and measured.Results:?1?The transfection efficiency rate was the highest when mice fibroblasts cells were transfected by adenovirus with multiplicity of infection?MOI?=100.RT-qPCR and ELISA results showed the fibroblasts cells transfected by AD-EGFP-VEGF₁₆₅,expressed effectively VEGF after 72h?p<0.05?.ELISA was used to detect the change of VEGF protein in the supernatant of cells cultured for 1 week.The results showed that VEGF protein reached its peak at 6 days.MTT results showed that there was no significant difference in the absorbance values of cells between the transfection group(AD-EGFP-VEGF₁₆₅)and the non-transfected group?p<0.05?.?2?After 20 weeks of feeding with arecoline,OSF-like changes were observed in the cheek tissues of mice in the experimental group.?3?After buccal submucosa tissue injection for 6 days,VEGF was effectively expressed in buccal submucosal tissues of OSF mice in AD-EGFP-VEGF₁₆₅group?p<0.05?,while no positive expression observed in AD-EGFP group and normal saline group.The number of microvessels detected by CD34 staining in the AD-EGFP-VEGF₁₆₅ group was significantly higher than that in the control group and AD-EGFP group?p<0.05?.Conclusion:?1?When the MOI value is 100,fibroblasts cells transfected by AD-EGFP-VEGF₁₆₅ can effectively express VEGF without affecting cell biological characteristics.?2?After 20 weeks of feeding with arecoline drinking method,OSF-like changes can be observed in the cheek tissues of mice in the experimental group.?3?AD-EGFP-VEGF₁₆₅ transfecting buccal submucosa tissue of OSF mice can effectively express VEGF and promote local angiogenesis.