Construction ANG-shRNA Lentivirus Vector And Its Effect On Rat With Diabetic Retinopathy

Objective The model of type 1 diabetic rats was established by streptozotocin(STZ).And the ANG-shRNA lentiviral vector contructed was used to interfere with the expression of ANG mRNA in retina of diabetic rats to reveal the effect of ANG on diabetic retinopathy.Methods ANG-shRNA lentiviral vector was constructed and screened the highest efficient shRNA sequence by acting on rat hepatocellular carcinoma(RH-35)cells and diabetic retinas of rats to interfer the expression of ANG mRNA in retina of diabetic rats.The structural changes of retinal vessels and retinal cells in diabetic rats were observed by PAS staining and electron microscopy.The expressions of ANG,VEGF and related factors Bcl-2,Bax,Hsp27 were detected by real-time quantitative PCR(q PCR)and Western blot.Results 1 ANG-shRNA-2 was selected as the most efficient sequence by in vitro and in vivo experiments.2 The model of type 1 diabetes rats was established successfully by STZ.The incidenct of model was 92.9%.3 The ratinas in the interfered rat group were observed by Evans blue in vivo perfusion of the retina,the distribution of blood vessel tended to normal,and the phenomenon of leakage and oozing was decreased.4 PAS staining showed that the inner limiting membrane of the rat retinas in the interference group was smooth and the number of cells in each layer was significantly increased.5 Electron microscopy showed that the vascular lumen of the interefered group was more complete,the chromatin of the rod cells was well-distributed in the nuclus,the vacuolar degeneration of mitochondria was reduced,the membrane shape tended to normal and arranged neatly.6 The relative expression of ANG,VEGF,Bcl-2,Bax and Hsp27 was detected by q PCR and Western blot.The relative expression of ANG,VEGF and Bax in interfered group was significantly lower than that in NC group,the expression of ANG,VEGF and Bax in the other groups was significantly higher than that in the negative control group(P <0.05).The expression of Bcl-2 in the interference group was higher than that in the NC group.The expression of Hsp27 in the interfered group,NCgroup and model group was increased.And except for negetive control,the expression of Bcl-2 and Hsp27 were significantly increased with the prolongation of the course of disease,and the differences were statistically significant(P <0.05)Conclusion ANG-shRNA lentiviral vector,constructed and acted on the retina of type 1 diabetic rats,reduced the expression of ANG mRNA in the retina and had a certain inhibitory effect on the diaberic retinopathy.