The Correlation Study Between The Expression Of MiR-19a Mediated By Adeno-associated Virus And The Formation Of Pulmonary Arterial Hypertension In Rats

To build the rat model with pulmonary arterial hypertension by left pneumonectomy.Find a way to transfect miR-19a into the pulmonary artery.To research the effect of differential expression of miR-19a on the severity of pulmonary revascularization and pulmonary hypertension in rats,and clarify the regulatory relationship of miR-19a,VEGF and BMPR2,so as to explore a potential targets and means for diagnosis and treatment for pulmonary arterial hypertension.objective:1)Perform the left pneumonectomy on SD rats to establish the pulmonary hypertension model.Transfect the adeno-associated virus which is mediated the target gene into the pulmonary artery of rats through the tracheal catheter.After three weeks feeding,use the fluorescence microscopy to observe the expression of green fluorescent protein in pulmonary artery after fast frozen sections,use qRT-PCR to detect miR-19a content at eighth weeks;2)30 6-8 weeks old SD rats were randomly divided into 5 groups,6 rats in each group,group C was sham operation group,group P0 group underwent left pneumonectomy;group P1underwent left pneumonectomy,and use the tracheal catheter to transfect adeno-associated virus?AAV?mediated over expression of miR-19a as soonn as the surgery operation was over,the virus titer was 1*10¹² v.g./ml;group P2underwent left pneumonectomy,and then use the tracheal catheter to transfect the same amount of adeno-associated virus?AAV?mediated miR-19a sponge;group P3 underwent left pneumonectomy,and then use the tracheal catheter to transfect the same amount of airborne AAV.After 8 weeks of feeding,measuring the mean pulmonary arterial pressure?mPAP?,and each group were sacrificed animal lung tissue HE staining,observe pathological changes of small pulmonary vessels.Detect small pulmonary vascular wall,pulmonary vascular diameter,and the ratio of vascular wall thickness and vessel diameter?MT%?under the microscope,so as to judge the lung small vessel muscularization.TheBMPR2expressionweredetectedby immunohistochemistry;and the expression level of miR-19a and vascular endothelial growth factor?VEGF?mRNA were detected by qRT-PCR in lung tissue.Use the Western-blot to measur the expression level of BMPR-2 protein in lung tissue.Methods:1)established the rat model of pulmonary hypertension by left pneumonectomy,after eighth weeks,all of the pneumonectomy group showed that the mean pulmonary arterial pressure?mPAP?and the muscularization of small pulmonary vessels were increased.The medial smooth muscle become thickened and hypertrophy,endothelial cell proliferated and its structure becomed disorder;3 weeks after transfection of adeno-related virus,we observed that the marked enhanced green fluorescent protein?eGFP?was expressed in the pulmonary artery under the inverted fluorescence microscope.2)The mean pulmonary arterial pressure and MT%in operation group which including group P0?group P1?group P2 and group P3 were higher than those in normal control group C,the difference was statistically significant?p<0.05?.while in the virus group which including group P1?group P2 and group P3,relative to the P3 group,the mean pulmonary arterial pressure and MT%was increased significantly in group P1,and in group P2 they were decreased significantly,there was statistically significant difference among group P1,group P2 and group P3?p<0.01?;the immunohistochemical staining of BMPR? in pulmonary artery was showed as?+++?,operation group was significantly decreased,and especially there were almost no staining?-?showed in group P1;compared with group C,the expression level of VEGFmRNA and miR-19a was increased in group P0,group P1,and group P3;they were significantly increased in group P1 compared to those in all group,and they were decreased in group P2 compare to those group P1 and P3?p<0.01?;the expression level of BMPR-2 protein were decreased significantly in group P0 and P1;they were decreased most significantly in P1 group and increased significantly in group P2.There was no significant difference between the indexes of P0 and P3?p>0.05?.Conclusion:1.Through the respiratory tract,the recombinant adeno-associated virus type 6?rAAV6?carrying the target gene was transfected into the rat pulmonary artery,and the target gene could be expressed continuously and efficiently.It can be used for targeted treatment of pulmonary hypertension and the treatment of related pulmonary diseases.2.The pneumonectomy model in rats can be used to simulate the pathophysiological status of high flow pulmonary hypertension and total pneumonectomy,and provide a research platform.3,miRNA19a regulates the expression of BMPR2 protein and the expression of VEGF to affect the vascular remodeling of pulmonary hypertension.