Construction and effect of recombinant adenovirus vector containing siRNA targeting Raf-1 on cardiomyocyte hypertrophy in neonatal rats
10.3969/j.issn.1671-7856.2016.04.004
- VernacularTitle:Raf-1基因重组腺病毒 siRNA 载体构建及其对大鼠心肌细胞肥大的影响
- Author:
Yaping ZHENG
;
Chunjie LIU
- Publication Type:Journal Article
- Keywords:
Raf-1;
siRNA;
Recombinant adenovirus vector;
Cardiomyocytes;
Rat
- From:
Chinese Journal of Comparative Medicine
2016;26(4):18-23
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct an adenovirus vector expressing small interfering RNA ( siRNA) targeting to rat Raf-1 gene and identify its function in cardiomyocytes.Methods The siRNA containing DNA sequence targeting to Raf-1 and its negative control sequence were designed, synthesized, annealed and subcloned into adenoviral shuttle vector pAdTrack-CMV.The recombinant adenovirus vector pAd-siRaf-1 was obtained by homologous recombination with pAdTrack-siRaf-1 linearized by PmeI and pAdeasy-1 in bacteria BJ5183, then transfected into HEK293 cells to package the adenovirus.Cardiomyocytes were infected with the adenovirus pAd-siRaf-1, and the expressions of Raf-1 and NF-κB protein were detected by Western blotting.[ 3 H ]-leu incorporation was evaluated by scintillation.The surface area of cardiomyocytes was measured using a HJ2000 image analysis system.Results The adenovirus vectors were verified by enzyme digestion and DNA sequencing.Compared with the Ang II group, Raf-1 and NF-κB expression, the surface area and [ 3 H]-leu incorporation of cardiomyocytes were significantly decreased in cardiomyocytes infected with the adenovirus PAd-siRaf-1.Conclusions A recombinant adenovirus vector containing rat siRaf-1 gene is successfully constructed.It can effectively reduce Raf-1 and NF-κB expression and cardiomyocyte hypertrophy induced by Ang II.
