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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 recombinant adeno-associated virus vector (AAV) has a number of safety-related features that facilitate its therapeutic use in the treatment of atherosclerosis.However, the traditional treatment of single genes is less than ideal because of the atherosclerosis In order to overcome this problem, a recombinant adeno-associated virus (AAV) -mediated gene therapy for atherosclerosis was studied.The experimental rats were divided into two groups: atherosclerosis model AAV-apoAⅠ / SR-BⅠ, AAV-apoAⅠand AAV-GFP were injected into the tail vein of rats and the normal diet group (normal control group) respectively. The rats in the atherosclerotic group were randomly divided into three groups. The tail vein was injected with PBS as a control, the mRNA of the target gene was detected by RT-PCR, the protein expression was detected by Western blotting and ELISA, and induced by diet-induced atherosclerosis and hypercholesterolemic rats were frozen 8 weeks after tail vein injection The fluorescence intensity of the recombinant AAV vector was detected by flow cytometry.After 8 weeks of intravenous injection of tail vein, the total plasma concentrations of AAV-apoAⅠ / SR-BⅠ and AAV-apoAⅠ And low-density lipoprotein cholesterol levels were significantly decreased compared with those of AAV-GFP treatment group (P <0.05), there was no significant difference between high-density lipoprotein cholesterol levels in each group, color Doppler ultrasound examination found that AAV-apoA Ⅰ Compared with AAV-GFP treatment group, the intima-media thickness of the abdominal aorta in the SR-BⅠ and AAV-apoAⅠgroups decreased significantly (P <0.05) and the levels of serum hs-CRP and SOD increased significantly (P <0.01) .At the same time, the expression of atherosclerosis-related gene mRNA was also detected.The results showed that after rAAV-hapoAⅠ-IRES-hSR-BⅠ treatment, the level of serum MDA decreased obviously The activity of NF-κB exerts an anti-inflammatory effect that reduces the release of inflammatory factors, increases the stability of atherosclerotic plaques, and decreases plasma cholesterol levels.In summary, the use of type 2 adeno-associated virus vector-mediated gene transfer overexpression of human apolipoprotein AⅠ And SR-BⅠhad beneficial effects on diet-induced hypercholesterolemia and atherosclerosis in rats.All these results may provide a new idea for the gene therapy of atherosclerosis.