摘要
目的探究机械敏感性离子通道Piezo1的shRNA干扰载体对ApoE缺陷小鼠动脉粥样硬化(AS)形成作用机制的实验研究。方法构建shRNA Piezo1的siRNA的干扰载体,慢病毒转染人胚肾293T细胞,RT-PCR和Western Blot筛选最佳干扰序列。选取健康的雄性昆明小鼠10只和ApoE基因缺失[ApoE(-/-)]小鼠30只,分成4组:空白对照组,ApoE(-/-)AS模型组,ApoE(-/-)阴性对照组,shRNA-Piezo1干预组。利用HE染色,油红染色观察AS的斑块。利用RT-qPCR、Western-Blot和免疫组化等方法检测生长因子血管内皮生长因子(VEGF)和成纤维细胞生长因子2(FGF-2)的表达。结果慢病毒转染结果显示,10^(-1)μl慢病毒量转染率最高,病毒滴度为1×10^(8) TU/ml。RT-PCR和Western Blot检测结果显示,shRNA-Piezo1-homo-5762组的mRNA水平要明显低于shRNA-Piezo1-homo-1875组和shRNA-Piezo1-3299组,比较具有统计学差异(P<0.05);说明shRNA-Piezo1-homo-5762为有效干扰序列。HE染色和油红染色结果显示,shRNA-Piezo1干预组中主动脉粥样硬化斑块体积所占总体积的百分比与ApoE(-/-)AS模型组比,体积明显缩小,具有统计学差异(P<0.05)。PT-PCR和Western blot结果显示,shRNA-Piezo1干预组中主动脉VEGF和FGF-2表达量明显低于AS模型组,具有统计学差异(P<0.05)。结论ShRNA-Piezo1通过抑制生长因子VEGF蛋白和FGF-2蛋白的表达发挥其抑制AS的作用。
Objective To investigate the effective mechanism of shRNA interference vector of mechanical sensitive ion channel Piezo1 in formation of atherosclerosis(AS)in ApoE deficient mice.Methods ShRNA interference vector of shRNA Piezo1 was established,and lentivirus was transfected into 293T cells of human embryonic kidney.The best interference sequence was screened by using RT-qPCR and Western blotting assay.Healthy male Kunming mice(n=10)were chosen into blank control group,and ApoE deficient mice(n=30)were divided into AS model group,negative control group and shRNA-Piezo1 interference group(each n=10).The plaques of atherosclerosis were observed after HE staining and oil red staining.The expressions of vascular endothelial growth factor(VEGF)and fibroblast growth factor-2(FGF-2)were detected by using RT-qPCR,Western blotting assay and immunohistochemistry technique.Results The results of lentivirus transfection showed that 10^(-1)μl of lentivirus had the highest transfection rate,and viral titer was 1×10^(8) TU/ml.The results of RT-qPCR and Western blotting assay detections that mRNA level was significantly lower in shRNA-Piezo1-homo-5762 group than those in shRNA-Piezo1-homo-1875 group and shRNA-Piezo1-3299 group(P<0.05),which indicated that shRNA-Piezo1-homo-5762 was an effective interference sequence.The results of HE staining and oil red staining showed that the percentage of aortic atherosclerotic plaque volume in the total volume was significantly smaller in shRNA-Piezo1 interference group than that in AS model group(P<0.05).The results of PT-qPCR and Western blotting assay showed that expressions of VEGF and FGF-2 were significantly lower in shRNA-Piezo1 interference group than those in AS model group(P<0.05).Conclusion ShRNA-Piezo1 plays a role of restraining AS through inhibiting the expressions of VEGF and FGF-2.
作者
王秀玲
王正忠
Wang Xiuling;Wang Zhengzhong(School of Clinical Medicine,Department of Medicine,Qingdao University,Qingdao 266000,China;不详)
出处
《中国循证心血管医学杂志》
2022年第3期345-349,355,共6页
Chinese Journal of Evidence-Based Cardiovascular Medicine
