摘要
目的探讨视蛋白在人皮肤微血管内皮细胞中的表达,及其对皮肤微血管内皮细胞的增殖、迁移及小管形成的影响。方法通过实时荧光定量PCR和蛋白质印迹法分别验证视蛋白的基因表达水平和蛋白表达水平。然后使用小干扰RNA技术沉默视蛋白3,分别用实时荧光定量PCR和蛋白印迹法验证视蛋白3的沉默效率。小干扰RNA成功沉默视蛋白3后,通过CCK-8法检测细胞的增殖活力,Transwell检测细胞的迁移能力,小管形成实验评估血管形成能力的情况。最后通过蛋白印迹法检测信号通路相关蛋白的表达情况。结果视蛋白1~5均在血管内皮细胞中表达,其中视蛋白3基因表达水平较其他视蛋白表达高,差异有统计学意义(P<0.05);沉默人皮肤微血管内皮细胞中视蛋白3后,细胞的增殖、迁移、小管形成能力均减弱,差异有统计学意义(P<0.05),血管生成的关键蛋白血管内皮生长因子受体2、磷脂酰肌醇3-激酶/蛋白激酶B、细胞外调节蛋白激酶1/2、内皮型一氧化氮合酶的表达均降低。结论视蛋白3在人皮肤微血管内皮细胞中高表达,并调控血管形成,可能成为血管生成相关疾病的一个潜在治疗靶点。
Objective To explore the expression of opsins in human skin microvascular endothelial cells and their effect on proliferation,migration and tube formation.Methods The mRNA levels of opsins in endothelial cells were detected by realtime PCR.The protein levels of opsins were detected by Western blot.Then,opsin3 knockdown using small interfering RNA technology was performed,and real-time PCR and Western blot were used to detect the silencing efficiency.After successfully silencing opsin3 by small interfering RNA,cell proliferation activity was detected by CCK-8 assay,cell migration ability was detected by Transwell assay,and angiogenesis ability was evaluated by tube formation assay.Finally,the activation of signaling pathways was analyzed by Western blot.Results All opsins(1~5)were expressed in endothelial cells,and the expression levels of opsin3 were higher than the others(P<0.05).The expression of VEGFR-2,PI3K/AKT,ERK1/2 and eNOS,which are key proteins in angiogenesis,was decreased after opsin3 silencing in endothelial cells(P<0.05).Conclusion Opsin3 is highly expressed in endothelial cells and regulates angiogenesis.Opsin3 may be a potential target for the treatment of angiogenesis-related diseases.
作者
罗欢欢
兰应华
曾雯
董仙
汪宇
陆洪光
LUO Huanhuan;LAN Yinghua;ZENG Wen;DONG Xian;WANG Yu;LU Hongguang(Guizhou Medical University,Guiyang 550025,China;Department of Dermatology,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China)
出处
《中国皮肤性病学杂志》
CAS
CSCD
北大核心
2021年第7期740-745,757,共7页
The Chinese Journal of Dermatovenereology
基金
国家自然科学基金(81972920,81673069)
国家临床重点专科建设项目(皮肤科),卫办医政函[(2012)649号]。
