R65 Ribozyme Gene Via Adeno-associated Virus Serotype 9 Transfection For Prevention Of Atherosclerosis

Objective:Recombinant adeno-associated virus serotype 9 as the carrier mediated R65 ribozyme gene transduction of ApoE-/- mice, observe whether the expression pathway can inhibit NF-κB signal aorta, thereby reducing inflammation, prevention the occurrence of atherosclerosis. Methods:(1) 8-week-old ApoE-/- mice were given high-fat diet and ordinary diet for 8,12,16,20,24 weeks later, lipids expression was detected, the expression of plaque area by oil red O staining, Western blot detection of high-fat diet aortic expression of NF-κB signaling pathway of P65 and P50. (2) 8-week-old ApoE-/ mice fed high-fat diet for 16 weeks, in 14,21,28,35,60 day aortic tissue specimens, laser scanning confocal microscope and Western blot to detect the expression of eGFP aorta. (3) 8 week old ApoE-/- mice tail vein injection of rAAV9-eGFP and rAAV9-eGFP-R65, while giving high fat diet, blood lipid levels were determined after 16 weeks, gross oil red O staining plaque area expression, expression of Western blot detection of NF-κB signaling pathway in P65 and P50, ELISA to detect the expression of inflammatory factor TNF-α and IL-6. Results:(1) Lipid test results showed that high-fat diet group, total cholesterol (TC), triglyceride (TG) and low density lipoprotein (LDL-C) levels were significantly higher than the ordinary diet group, oil red O staining showed that high-fat diet group plaque lesions faster than normal diet group, and can effectively activate NF-κB signaling pathway. (2)The laser scanning confocal microscope and Western blot results showed that rAAV9-eGFP can transfect vascular, the peak eGFP expression in the first 35 days. (3) Serological and oil red O staining showed expression of transduced rAAV9-eGFP-R65 reduce lipid levels and plaque area. Western blot showed that after 16 weeks high fat diet can effectively activate NF-κB signaling pathway, and transfected rAAV9-eGFP-R65 can inhibit NF-κB signaling pathway activity. ELISA results showed that after rAAV9-eGFP-R65 transfection can effectively reduce the inflammatory cytokines TNF-a and IL-6 expression. Conclusion:High-fat diet can speed up the process of arterial pathology of atherosclerosis, and effectively activate NF-κB signaling pathway, can form a stable model of atherosclerosis at 16 weeks. rAAV9-eGFP be effective, long-lasting and stable transfection aortic atherosclerosis in ApoE-/- mice. rAAV9-eGFP-R65 can reduce the expression of activated NF-κB signaling pathway in the aorta, effectively reduce the progression of atherosclerosis.