摘要
目的构建特异性抑制大鼠Raf-1基因的重组腺病毒载体,并将其体外转导大鼠心肌细胞中进行功能鉴定。方法合成针对大鼠Raf-1的靶序列及阴性对照序列,经退火形成的DNA双链定向克隆到穿梭质粒p Ad Track CMV中获得p Ad Track-siRaf-1质粒,Pme I线性化后在BJ5183细菌中与p Ad Easy-1骨架质粒进行同源重组获得p Ad-siRaf-1质粒,后转染HEK293细胞,包装获得p Ad-siRaf-1腺病毒颗粒,继而感染原代培养的心肌细胞,通过Western blot方法检测siRNA对Raf-1、NF-κB基因的抑制效率,液体闪烁计数仪测定3H-亮氨酸([3H]-leu)掺入率,用HJ2000图像分析系统测定细胞表面积。结果重组腺病毒载体经酶切、鉴定正确,制备的病毒感染效率高,携带Raf-1的病毒颗粒能够在蛋白水平有效抑制Ang II诱导心肌细胞Raf-1的表达、细胞表面积及[3H]-leu的增加并下调Raf-1、NF-κB表达。结论成功构建了p Ad-siRaf-1重组腺病毒载体,并在HEK293细胞中包装成重组腺病毒,转染心肌细胞后能有效抑制Raf-1、NF-κB表达,抑制Ang II诱导的心肌细胞肥大。
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 p Ad Track-CMV. The recombinant adenovirus vector p Ad-siRaf-1 was obtained by homologous recombination with p Ad Track-siRaf-1 linearized by Pme I and p Adeasy-1 in bacteria BJ5183,then transfected into HEK293 cells to package the adenovirus. Cardiomyocytes were infected with the adenovirus p Ad-siRaf-1,and the expressions of Raf-1 and NF-κB protein were detected by Western blotting. [3H]-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 [3H]-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.
出处
《中国比较医学杂志》
CAS
北大核心
2016年第4期18-23,共6页
Chinese Journal of Comparative Medicine
基金
河南省教育厅青年骨干教师资助项目(No.2013GGJS-271)
