重组腺病毒载体敲减ApoE-/-小鼠血管平滑肌细胞Orai1基因模型的构建

Establishment of Orai1 gene knock down model in ApoE mouse vascular smooth mucsle cells with adenovirus interference vector

目的:为探讨Orai1基因在动脉粥样硬化进展中的作用,通过重组腺病毒载体建立ApoE-/-小鼠血管平滑肌细胞(VSMCs)Orai1基因的敲低模型,为深入相关研究奠定基础。方法:构建针对小鼠Orai1基因的重组腺病毒载体。高脂饲养ApoE-/-小鼠至16周龄后处死。取胸主动脉原代VSMCs改为体外培养,细胞分为三组,分别为shOrai1组、shNC组和对照组。转染后通过实时定量聚合酶链式反应(qPCR), Western blot检测Orai1表达水平,同时对转染模型进行验证。结果:qPCR示,重组腺病毒在ApoE-/-小鼠VSMCs中Orai1基因的干扰效率达57%,Western blot结果示,Ad-GFP-Orial-shRNA转染组Orai1蛋白表达水平显著降低。结论:通过成功建立ApoE-/-小鼠VSMCs中Orai1基因敲低模型,从而为阐明钙库操纵性钙通道在动脉粥样硬化进程中的作用,尤其关于斑块延展和不稳定的相关性研究奠定了基础。

Objective: To further investigate the role of calcium release-activated calcium channel modulator 1(Orai1) in the process of Atherosclerosis, the recombinant adenovirus vector was used to establish the ApoE-/- mouse vascular smooth mucsle cells(VSMCs) Orai1 gene knock down model for further related studies. Methods: A recombinant adenovirus vector(Ad-GFP-Orial-shRNA) targeting the Orai1 gene in mice was constructed. ApoE-/- mouse fed with high-fat diet were sacrificed at 16 weeks of age. VSMCs extracted from the thoracic aorta were cultured in vitro. The cells were randomly divided into 3 groups for Ad-GFP-Orial-shRNA infection, Ad-GFP-NC-shRNA infection and control group. Orai1 expression was tested by qPCR(quantitative real-time polymerase chain reaction) and Western blot to validated the model. Results: qPCR showed that the Orai1 mRNA in VSMCs decreased by 57%, Western blot showed that Orai1 protein expression level was significantly reduced. Conclusions: The ApoE-/- mouse VSMCs Orai1 gene knock down model was successfully established, building a foundation to further study the mechanism of SOCC participate in the process of atherosclerosis and its impact on plaques’ extension and instability.