摘要
重组腺相关病毒载体(AAV)具有很多安全方面的特性,有利于其在动脉硬化方面的治疗研究.尽管如此,传统的介导单个基因的治疗效果并不是很理想,这都归因于动脉硬化疾病的发生是由于多种基因的缺陷而不是单单某一特定基因.为了克服这个问题,尝试了重组腺相关病毒载体介导的双基因对动脉硬化的治疗研究.实验大鼠分为动脉硬化模型鼠和正常饮食组(即正常对照组),动脉硬化组大鼠被随机分为3组,分别进行AAV-apoAⅠ/SR-BⅠ,AAV-apoAⅠ,与AAV-GFP尾静脉注射,同时正常饮食组尾静脉注射PBS作为对照.其中目的基因mRNA检测采用RT-PCR方法,蛋白质表达的检测采用Western blotting和ELISA.由饮食诱导的动脉硬化和高胆固醇的大鼠模型在尾静脉注射后8周进行冰冻切片的荧光检测.重组AAV载体显示出较强的表达活性.尾静脉注射治疗8周后,AAV-apoAⅠ/SR-BⅠ与AAV-apoAⅠ治疗组血浆总胆固醇和低密度脂蛋白胆固醇浓度与AAV-GFP治疗组相比有了明显下降(P<0.05),高密度脂蛋白胆固醇浓度各组之间没有明显差异,彩色多普勒超声检测发现,AAV-apoAⅠ/SR-BⅠ与AAV-apoAⅠ治疗组的腹主动脉的内中膜厚度相对于AAV-GFP治疗组有了明显下降(P<0.05),血清hs-CRP和SOD的水平有了明显上升(P<0.01),血清MDA的水平有了明显的下降(P<0.01).同时也检测了动脉硬化相关基因mRNA水平的表达.结果显示,rAAV-hapoAⅠ-IRES-hSR-BⅠ治疗后可能是通过抑制NF-κB的活性发挥抗炎作用减少炎症因子的释放,增加动脉硬化板块的稳定性以及降低血浆胆固醇含量的.总之,利用2型腺相关病毒载体介导的基因转移过度表达人载脂蛋白AⅠ和SR-BⅠ可能对饮食诱发大鼠高胆固醇血症和动脉硬化的产生有利影响.这些结果可能为动脉粥样硬化基因治疗提供了一种新的研究思路.
The adeno-associated virus （AAV） has many safety features that favor its use in the treatment of arteriosclerosis; however, the conventional, adeno-associated virus （AAV） mediated single-gene delivery is inefficient for arteriosclerosis. This has been attributed that the incidence of atherosclerosis is caused by a variety of genetic defects but not a particular gene. To overcome this, double-gene delivery was evaluated for the treatment of atherosclerosis. Four experimental groups were administered the following AAV vector constructs： rAAV-apoA I -IRES-SR-B I , rAAV-apoA I -GFP, rAAV-IRES-GFP, and PBS. ApoA I and SR-B I gene expression was detected using RT-PCR. The apoA I and SR-B I protein expression was determined by Western blotting and ELISA. Diet-induced hypercholesterolemia and atherosclerosis in rats was adopted and rAAV was administered through the tail vein injection. HepG2 cells were cultured and infected with the three viral vectors. The apoA I and SR-B I secreted from HepG2 cells in the AAV- apoA I/SR-B ｜ group enhanced cholesterol effiux and resulted in a stronger RCT ability, respectively. In the rats＇ model with diet-induced hypercholesterolemia and atherosclerosis, GFP expression could be detected at 8 weeks post-injection. The rAAV vector had superior gene expressing activity. Eight weeks after gene transfer, plasma total cholesterol and LDL-cholesterol concentrations were significantly reduced （P 〈 0.05） compared to control for rAAV-IRES-GFP （AAV-GFP） treated group. No effect on HDL-cholesterol concentrations occurred. Ultrasound determined intima-media thickness also has been significantly reduced compared to control. Serum hs-CRP and SOD levels increased significantly （P 〈 0.01）. Serum MDA levels decreased significantly. Gene mRNA expression was detected in atherosclerosis rats＇ model. The results show that rAAV-hapoA I-IRES-hSR-B I vector can anti-inflammatory, reduce atherosclerotic macrophage content and increases lesion stability of pre-existing plaques thr
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2011年第9期830-841,共12页
Progress In Biochemistry and Biophysics
基金
supported by a grant from Shenzhen Institute of Xiangya Biomedicine~~
关键词
2型腺相关病毒载体
载脂蛋白A
I
B族I型清道夫受体
胆固醇逆向转运
基因治疗
adeno-associated virus, apolipoprotein A- I , scavenger receptor class B type I , reverse cholesterol transport （RCT）, genetherapy
