Abstract:Aim To construct and identificate recombinant adenovirus with siNrdp1 gene using AdMax system, and investigate the effect of Nrdp1 gene on cardiomyocyte hypertrophy. Methods Designing and synthesizing siRNA sequences targeting of Nrdp1 DNA, then cloned into the shuttle vector GV119 and homologous recombinated with adenovirus backbone plasmid AdMax in BJ5183 bacteria transfected HEK293 cells, and got adenovirus containing Nrdp1-siRNA gene through packaging. Real-time quantitative PCR and Western blot were used to detect Nrdp1 expression in primary rat neonatal cardiomyocytes. After adenoviral containing siNrdp1 transfection and angiotensin Ⅱ （AngⅡ） stimulation, real-time quantitative PCR was used to detect the expression of myocardial hypertrophy marker gene （ANF, β-MHC and Skeletal-α-actin） of rat neonatal cardiomyocytes. Results Digested PCR analysis and sequencing showed that interference Nrdp1 adenovirus was successfully constructed, and the titer of virus was 1.5E＋9 PFU/mL. Real-time PCR and Western blot indicated that the expressions of Nrdp1 mRNA and protein were greatly inhibited after infection in rat primary cardiomyocytes with recombinant adenovirus particles （P<0.001）. Nrdp1 gene silencing cloud significantly increase expression of AngⅡ induced cardiomyocyte hypertrophy marker genes including ANF, β-MHC and Skeletal-α-actin （P<0.01）. Conclusion The recombinant adenovirus vector containing the Nrdp1-siRNA gene was successfully constructed, which can effectively silence Nrdp1 gene and enhance AngⅡ induced cardiomyocytes hypertrophy in vitro.

Abstract:Aim To construct an adenovirus expression vector which can express hepatocyte growth factor (HGF) in vascular smooth muscle cells (SMC). Methods The plasmid containing HGF fragment was cleaved by restriction enzyme digestion, and the resultant fragment was inserted directionally into adenoviral shuttle plasmid. The linearized recombinant adeno viral shuttle plasmid and adenovirus expression vector were cotransformed into Escherichia coli BJ5183 cells for homologous recombination . The resultant recombinant plasmid, pAd-HGF, then was transfected into HEK293 cells with liposome for packaging. The recombinant adenoviral shuttle plasmid and pAd-HGF were identified by enzyme digestion and sequencing. The package of pAd-HGF in HEK293 cells was tracked by fluorescent microscope, and was observed by electronic microscope. The expression of packaged pAd-HGF in abdominal aortic SMCs of rat was identified by RT-PCR and Western blotting. Results HGF fragment was inserted correctly into the adenoviral shuttle plasmid and adenovirus expression vector. High-liter packaged adenovirus vector was produced and expressed in abdominal aortic SMCs of rat. Conclusions A recombinant adenovirus expression vector of HGF was constructed successfully. This study suggested that HGF may be a potential target for the gene therapy of vascular diseases and established a foundation for further study.

Abstract:Aim To explore the possibility of endothelial nitric oxide synthase (eNOS)gene transfer preventing restenosis of vein grafts. Methods We constructed the recombinant adenovirus vector that coding eNOS, AdCMV eNOS and adenovirus vector (AdCMV). Artery bypass animal model was constructed: we took jugulars of goat as grafts, infected AdCMV eNOS and AdCMV in vitro, then anastomosed the vein grafts between carotids of goats. The functional expression of eNOS in vein grafts was assessed using immunohistochemical staining and measurement of NO concentration. The inhibition of intimal hyperplasia in vein grafts transducted AdCMV eNOS was assessed using assay of 3 H-TDR incorporation, histologic analysis, measursment of intimal thickness and area percentage of stenosis of vein grafts. Results Expression of eNOS gene in vein grafts began on 48 h after transducted with AdCMV eNOS. Levels of NO in vein grafts: AdCMV eNOS transduced veins yielded 1724.1±312.2 μmol/(L·g ), whereas AdCMV yielded 245.8±17.4 μmol/(L·g) (p<0.001, n=3). Levels of 3 H-TDR Incorporation: Incorporation in AdCMV veins was 487.7±51.1 cmp/mg vessel, and 140.8±32.5 cmp/mg vessel in AdCMV eNOS veins (p<0.01, n=3). Mean thickness of intimal was 125.7±30.60 μm in AdCMV veins, and 28.8±5.24 μm in AdCMV eNOS veins; area percentage of stenosis was 52.18%±8.46% in AdCMV veins, and 23.6%±4.71% in AdCMV eNOS veins. Conclusions Adenovirus-mediated eNOS gene transfer could inhibit intimal hyperlasia effectively. This work lay a foundation in gene therapy of grafts restenosis after CABG.