血管扩张刺激磷蛋白对抵抗素促进冠状动脉内皮细胞增殖和迁移的影响

发布时间：2018-06-02 09:24

本文选题：抵抗素 + 血管扩张刺激磷蛋白　；参考：《医学研究生学报》2017年12期

【摘要】：目的抵抗素可通过导致内皮细胞功能的失调,参与动脉粥样硬化的发生和发展,然而其作用机制研究较少。文中旨在研究抵抗素对冠状动脉内皮细胞功能的影响,探讨影响抵抗素作用的可能机制。方法构建针对人血管扩张刺激磷蛋白(VASP)的慢病毒表达载体的VASP mRNA的shRNA干扰序列(LV-si VASP组)和阴性对照载体(LV-sicntr组),并将其感染高表达VASP的人冠状动脉内皮细胞,RT-PCR检测VASP mRNA的表达,Western blot测定蛋白的表达。用不同浓度的抵抗素(0、10、50、100、200 ng/m L)体外作用于人冠状动脉内皮细胞系(HCAECs),MTT法检测抵抗素对细胞增殖的作用,Transwell小室实验检测细胞迁移能力的改变。在相同抵抗素浓度100 ng/m L下,检测2组对内皮细胞增殖、迁移影响。免疫荧光法分析2组细胞表面受体血管内皮生长因子受体2(VEGFR2)表达情况。采用pull-down法分析2组细胞Rho A活性。结果LV-si VASP组RNA表达抑制率[(68.1±0.8)%]、VASP蛋白表达抑制率[(59.3±1.7)%]较LV-sicntr组(100%)明显降低(P0.05)。与0 ng/m L比较,50~200 ng/m L抵抗素作用48 h后,可以显著促进细胞的增殖(P0.05)。50~200 ng/m L抵抗素处理24 h后,HCAEC呈剂量依赖性促进细胞迁移(P0.05)。沉默VASP后,抑制了抵抗素对细胞增殖和迁移的作用(P0.05)。荧光显微镜观察结果显示,与LV-sicntr组比较,LV-si VASP组VEGFR2表达未见明显变化,而LV-si VASP组Rho A活性较LV-sicntr组减弱[(41.3±3.1)%vs 100%],差异有统计学意义(P0.05)。结论抵抗素呈浓度依赖性的促进冠状动脉内皮细胞增殖、迁移,敲除VASP对抵抗素功能的影响与减弱的Rho A活性有关。
[Abstract]:Objective resistin is involved in the occurrence and development of atherosclerosis by causing dysfunction of endothelial cells. The purpose of this study was to investigate the effects of resistin on the function of coronary endothelial cells and the possible mechanism of affecting resistin. Methods the shRNA interference sequence of VASP mRNA targeting human vasodilation-stimulated phosphoprotein (VASP) lentivirus expression vector was constructed in LV-si VASP group and negative control vector LV-sicntr. RT-PCR was used to detect the infection of human coronary artery endothelial cells with high expression of VASP. The expression of VASP mRNA was detected by Western blot. The effect of resistin on proliferation of human coronary artery endothelial cell line (HCAECs) was detected by using different concentrations of resistin in vitro. The effect of resistin on cell proliferation was detected by Transwell chamber assay. Under the same resistin concentration of 100 ng/m L, the effects of two groups on endothelial cell proliferation and migration were detected. The expression of vascular endothelial growth factor receptor 2 (VEGFR 2) was analyzed by immunofluorescence. The activity of Rho A was analyzed by pull-down assay. Results the inhibitory rate of RNA expression in LV-si VASP group [68.1 卤0.8%] was significantly lower than that in LV-sicntr group (59.3 卤1.7%). Compared with 0 ng/m L, resistin could significantly promote cell proliferation after 48 h treatment with resistin 50 ng/m L in a dose-dependent manner, and promote cell migration in a dose-dependent manner. Silencing VASP inhibited the effect of resistin on cell proliferation and migration. The results of fluorescence microscope showed that there was no significant change in VEGFR2 expression in LV-si VASP group compared with LV-sicntr group, but the activity of Rho A in LV-si VASP group was lower than that in LV-sicntr group [41.3 卤3.1)%vs 100%], the difference was statistically significant (P 0.05). Conclusion resistin promotes the proliferation and migration of coronary endothelial cells in a concentration-dependent manner. The effect of knockout of VASP on resistin function is related to the decreased activity of Rho A.
【作者单位】：南方医科大学金陵医院(南京军区南京总医院)心脏内科;
【基金】：中国博士后科学基金(2016M603062) 江苏省“六大人才高峰”资助项目(WS-078) 南京军区医学科技创新重点课题(09Z026) 江苏省博士后科研资助计划项目(1601153B)
【分类号】：R54

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