| ObjectiveTo explore a new way of CHF treatment by BNP.Ad-hBNP were constructed andCHF rats were also prepared for observing the distribution and the expression of hBNPgene in CHF rats and evaluating the therapeutic effect of BNP on CHF rats through thegene therapy in vivo.This may lead to elaborate the CHF treatment by BNP and itsmechanism and provide a reference of basic research for BNP in gene therapy.Methods1.The target gene hBNP were obstained by reverse transcription polymerasechain reaction from human heart.The product of RT-PCR were ligated with pUCm-Tvectors to form pUC-hBNP.After pUC-hBNP were digested with KpnI and SalI,thetarget fragments were subcloned into shuttle plasimid pAdTrack-CMV.The resultantplasmid pAdTrack-hBNP,after linearized by PmeI,were transformed intoE.coilBJ5183 including adenoviral backbone plasmid pAdEasy-1,then therecombinant plasmid pAdEasy-hBNP were constructed.The pAdEasy-hBNP wereconfirmed by BamHI and PacI,then by DNA sequencing.Recombinant plasmidpAdEasy-hBNP linearized by PacI were transfected into 293T cells.The recombinantadenovirus Ad-hBNP were packaged and amplified in 293T cells.Determin the titer ofpurified recombinant adenovirus and observe the shape of recombinant adenovirus.2.The CHF rats were prepared by abdominal aorta constriction to 0.6mm.After12 weeks,echocadiagraphy,hemodynamics parameters,radioimmunoassay of serumlevels of AgII and Ald,pathological HE staining and Masson staining,RT-PCRanalysis of the expression of myocardial collagen mRNA were all performed toevaluate the cardiac function of CHF rats.3.To observe the distribution and the expression of hBNP gene in CHF rats 24CHF rats were randomly divided into 4 groups for detecting the serum levels of hBNPwith ELISA,the expression of hBNP with RT-PCR and immunohistochemistry indifferent groups.To elaborate the CHF treatment by BNP and its mechanism 30 CHFrats were randomly divided into Ad-hBNP group,Ad-Track group,NS group and 10sham-operated rats as control group.After 4 weeks treatment,echocadiagraphy, hemodynamics parameters,radioimmunoassay of serum levels of AgII,Ald,ET,TNF-αand RT-PCR analysis of the expression of myocardial collagen mRNA wereperformed to evaluate the cardiac function and the ventricular remodeling.ResultAd-hBNP were constructed successfully and the titer of purified adenovirus was4.4275×1012VP/ml.Recombinant adenoviruses were showed as polyhedron shapeunder electron microscopy.CHF rats were prepared and CHF rats for the experiment invivo must show symptoms of CHF,at the same time the criterions of echocadiagraphywere LVESD＞0.32cm,VLEDD＞0.65cm,EF＜75% and FS＜40%.The expression ofhBNP gene were detected in CHF rats by intraperitoneal injection Ad-hBNP and itcontinued at least 7 days.By the treatment of Ad-hBNP,the LVPW,IVS,LVEDD andLVESD of the Ad-hBNP group were significantly smaller than the Ad-Track group andthe NS group.Compared with the Ad-Track group and the NS group,hemodynamicsshowed HR,LVEDP decreased and LVSP,+dP/dtmax and the absolute of-dP/dtmaxincreased,while the serum levels of AgII,Ald,ET and TNF-α,as well the expressionof myocardial collagen mRNA of CHF rats were reduced.Conclusion1.Ad-hBNP were constructed successfully by using Ad-Easy defective adenovirussystem.2.CHF rats were prepared for the experiment in vivo by abdominal aortaconstriction.3.Exogenous hBNP were effectively expressed in CHF rats.Compared with thehalf life of BNP,the prosses that at least continued 7 days were significantly extended.4.BNP improved the cardiac structure and function.It may be play an importantrole in ventricular remodeling by controlling the neuroendocrine and cytokines.The research showed that Ad-hBNP had a therapeutic role on CHF rats both incardiac function and ventricular remodeling and tried a new way of CHF treatment byBNP.It also provided a reference of basic research for BNP in gene therapy.
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