摘要
[目的]探讨小干扰RNA(siRNA)沉默人脐静脉血管内皮细胞(HUVECs)对组织蛋白酶S(CatS)表达的影响.[方法]利用Sigma公司设计合成的以CatS为靶标的siRNA,通过脂质体转染HUVECs.选取抑制率最高的siRNA转染HUVECs,分为空白对照组、阴性对照组(siLam)、RNACatS组1(siCatS1)和siRNACatS组2(siCatS2),采用Western Blot法检测CatS、组织蛋白酶K(CatK)、过氧化物酶增殖物激活受体-γ(PPAR-γ)、胰岛素受体底物-2(IRS-2)、磷酸化内皮一氧化氮合酶(p-eNOS)及血管内皮细胞增殖因子(VEGF)的表达水平;利用细胞迁移、浸润、增殖实验观察细胞数的变化;采用微管腔形成实验观察微管腔形成数目.[结果] siCatS1,siCatS2组CatS表达水平明显低于空白对照组(P<0.05),而CatK表达水平间差异无统计学意义(P>0.05).PPAR-γ,IRS-2,p-eNOS和VEGF的免疫印迹图和表达水平的定量分析结果显示,与空白对照组比较,siCatS1,siCatS2组PPAR-γ,IRS-2,p-eNOS和VEGF表达水平显著降低(P<0.01),浸润细胞数显著减少(P<0.001),但迁移细胞数间差异无统计学意义(P>0.05).与空白对照组比较,siCatS1,siCatS2组增殖细胞数及微管腔形成数目明显减少(P<0.01).[结论] siRNA可显著降低PPARγ,IRS-2,P-eNOS和VEGF的表达,细胞浸润、增殖和微管腔形成减少机制认为可能与PPARγ,IRS-2,P-eNOS和VEGF蛋白表达水平的降低有关系.
OBJECTIVE To explore the effects of siRNA silencing human umbilical vein endothelial cells(HUVECs) on the expression of cathepsin S(CatS). METHODS SiRNA targeted at CatS was designed and synthesized by Sigma company and transfected into HUVECs by liposome. SiRNA with the highest inhibition rate was selected to transfect HUVECs, they were divided into control group, negative control group(siLam), siRNA cathepsin S group 1(siCatS1) and siRNA cathepsin S group 2(siCatS2). Western Blot was used to detect the expressions of CatS, CatK, peroxidase proliferator-activated receptor-gamma(PPAR-γ), insulin receptor substrate-2(IRS-2), phosphorylated endothelial nitric oxide synthase(p-eNOS) and vascular endothelial cell proliferation factor(VEGF). Cell migration, infiltration and proliferation experiment were used to observe the changes of cell number. Microtubule formation experiment was used to observe the number of microtubule formation. RESULTS The expression of CatS in siCatS1 and siCatS2 group was significantly lower than that in the control group(P<0.05), however there was no statistically significant difference in the expression of CatK(P>0.05). Quantitative analysis of immunoblotting and expression levels of PPAR-γ, IRS-2, p-eNOS and VEGF showed that the levels of PPAR-γ, IRS-2, p-eNOS and VEGF in siCatS groups were significantly lower than those in the control group(P<0.01). The number of infiltrating cells in siCatS groups was significantly lower than that in the control group(P<0.001), but there was no significant difference in the number of migrating cells(P>0.05). Compared with the control group, the number of proliferation cells and the number of microtubule formation in siCatS groups were significantly reduced(P<0.01). CONCLUSION SiRNA could significantly reduce the expressions of PPAR-γ, IRS-2, P-eNOS and VEGF, the mechanisms of cell infiltration, proliferation and reduction of microtubule formation might be related to the decrease of PPAR-γ, IRS-2, P-eNOS and VEGF protein expression levels.
作者
李文浩
贾钟楠
李香
LI Wenhao;JIA Zhongnan;LI Xiang(Department of Cardiology, Affiliated Hospital of Yanbian University, Yanji 133000, Jilin, China)
出处
《延边大学医学学报》
CAS
2019年第2期86-90,共5页
Journal of Medical Science Yanbian University
基金
国家自然科学基金资助项目(81660240)
