Study On Pulmonary Hypertension Induced By Left-To-Right Shunt By Overexpression Of Adenoviral Mediated P53 Gene Of The Wild Type Transfer In Rats

Objective To investigate the feasibility of transferring p53 gene into lung tissue with recombinant replication-deficient adenovirus inserted in the wistar rat models of pulmonary hypertension induced by left-to-right shunt,study the expression of the desired gene in vivo, find if overexpression of desired gene can inhibit pulmonary hypertension, observe the change of proliferation and apoptosis of vascular smoothmuscle cells(VSMCs).Methods Getting the gene p53 by making the pCMVp53 incised with the BamHland NheI.Cloning the cDNA to the Pcmv5GFP;making it to be adjusted by CMVS.Then transferring it and the right-DNA of the adenovirus to the 293 cell.Detemining p53 by ELISA.The control virus Adeno-lacZ was constructed in the same way.48 wistar rats were divided randomly into 4 groups.GroupⅠ(n=10)was control group.GroupⅡ(n=13)was model group, which were established PH models using intubation tube between common carotid artery and external jugular vein.GroupⅢ(n=14)was Adeno-p53 transfection group,which were established PH models and transfected Adeno-p53 using windpipe Inhalation.GroupⅣ(n=11)was Adeno-lacZ transfection group,which were established PH models and transfected Adeno-lacZ using windpipe inhalation.After 16 weeks,every group were mearsured mRVP,mPAP by right-heart catheterize-tionand and caculated RVHI and RV/BW.HE stained were used to observe the architecture changes in lung arteriole and measured the external diameter and thickness of vessel wall of lung arteriole to calculate WT%.Western blot was used to assessment the expression of p53 gene product in groupⅢandⅣ.Labelled streptavidin-biotin(LSAB)was used to investigate the expression of PCNA of VSMCs,and TUNEL was used to detect the apoptosis of VSMCs.Calculate PI(proliferation index),AI(apoptosis index)and PI/AI.Results 1.The mRVP and mPAP in GroupⅡ,ⅢandⅣwere markedly higher than those in GroupⅠ(P＜0.05)and the RVHI and RV/BW were also demostrated statistically different(P＜0.05).The mPAP,mPVP and WT%in GroupⅢwere markedly lower than those in GroupⅡandⅣ(P＜0.05),but higher than those in GroupⅠ(P＜0.05).In GroupⅡ,ⅢandⅣ,muscular hypertrophy in the media of pulmonary artery and arteriole was evident, and cavity of the vessels became strictured.2.specific segment was showed by Western blot in groupⅢ,which means the desired gene expressed successfully.3.In GroupⅢ,PI was markedly lower than PI in GroupⅡandⅣ(P＜0.05), and AI was higher than any other group(P＜0.05),and PI/AI was markedky lower than groupⅡandⅣ(P＜0.05).Conclusion 1.adenovirus can successfully carry p53 of the wild type and transfect the lung tissue of PH model in rats,and full expression of p53.2.p53 of the wild type gene can inhibit the development of pulmonary hypertensive through inducing the apoptosis of VSMCs.