人类免疫缺陷病毒-1负调控因子通过调控糖原合成酶激酶-3β/β连环蛋白信号通路促进人类疱疹病毒8型病毒白细胞介素6诱导血管生成  

Human immunodeficiency virus-1 negative factor protein promotes human herpesvirus-8 viral interleukin-6-induced angiogenesis： role of glycogen synthase kinase-3β/β-catenin signaling pathway

作　　者：姚水洪[1] 邱惠萍[1] 刘建军[1] 朱小飞[2] 秦娣[3] 严沁[3] 卢春[3] 

机构地区：[1]浙江省衢州职业技术学院医学院,324000 [2]江苏省中医院检验科 [3]南京医科大学微生物学与免疫学系

出　　处：《中华传染病杂志》2014年第4期193-198,共6页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金资助项目（81171552）;浙江省教育厅科技计划项目（Y201328970）;浙江省教育厅高校访问学者教师专业发展项目（FX2012120）;衢州市科技计划项目（2013Y020）

摘　　要：目的 探讨糖原合成酶激酶（GSK）3β/β连环蛋白信号通路在HIV 1负调控因子（Nef）蛋白促进人类疱疹病毒8型（HHV-8）病毒白细胞介素6（vIL-6）诱导血管生成中的作用.方法 将GSK-3β突变质粒GSK-3β-S9A、显性负相质粒GSK 3β-DN及其对照质粒pcDNA3.1＋分别转染稳定表达HHV-8 vIL-6、HIV-1-Nef以及共表达vIL-6和Nef蛋白的内皮细胞,观察EA.hy 926细胞微管形成;将上述转染质粒的细胞接种鸡胚绒毛尿囊膜（CAM）检测血管生成情况;Western印迹法检测转染上述质粒的细胞及CAM组织中GSK-3β/β连环蛋白通路中信号分子的表达水平.计量资料两两比较采用t检验.结果 转染GSK-3β-DN降低GSK-3β活性,能增强HIV-1 Nef蛋白促进vIL-6诱导微管形成（3.42比2.51,t=3.67,P＜0.01）和血管生成（6.25比3.97,t=4.06,P＜0.01）的能力.转染GSK-3β-S9A活化GSK-3β,则能显著抑制Nef蛋白的上述功能（0.62比2.51,t=8.48,P＜0.01;0.39比3.97,t=8.59,P＜0.01）.转染GSK-3β-DN后,细胞或组织中信号通路下游β连环蛋白（细胞中：3.53比2.07,t=6.60,P＜0.05;组织中：2.76比1.74,t=17.40,P＜0.01）、血管内皮生长因子（VEGF,细胞中：2.68比1.87,t=4.28,P＜0.01;组织中：2.20比1.39,t=7.08,P＜0.01）的表达升高;转染GSK-3β-S9A后,GSK-3β的磷酸化水平降低（细胞中：0.50比1.47,t=7.33,P＜0.01;组织中：0.35比1.97,t=10.72,P＜0.01）,β连环蛋白（细胞中：1.05比2.62,t=29.50,P＜0.01;组织中：0.79比1.77,t=5.72,P＜0.01）、VEGF（细胞中：0.74比2.16,t=20.95,P＜0.01;组织中：0.43比1.65,t=11.89,P＜0.01）的表达也随之减少.结论 GSK-3β/β连环蛋白信号通路参与调控HIV-1 Nef蛋白促进HHV-8 vIL-6诱导血管生成,极可能成为临床治疗艾滋病患者卡波齐肉瘤的一个潜在分子靶点.Objective To explore the role of glycogen synthase kinase （GSK）-3β/β-catenin signaling pathway on human immunodeficiency virus 1 （HIV-1） negative factor （Nef） protein promoting of human herpesvirus-8 （HHV-8） viral interleukin-6 （vIL-6）-induced angiogenesis.Methods GSK-3β mutant plasmid GSK-3β-S9A,dominant negative （DN） form GSK-3β-DN and the control vector pcDNA3.1＋ were transfected into endothelial cells which stably expressed HHV-8 vIL-6 or HIV-1 Nef,or co-expressed vIL-6 and Nef protein.Microtubule formation assay was performed to explore microtubule formation ability.A chick embryo chorioallantoic membrane （CAM） model was used to detect angiogenesis.The expression of GSK-3β/β-catenin signaling pathway-related kinases in transfected cells and CAM tissue were further detected by Western blot.The measurement data were compared by t test.Results The activity of GSK-3β was decreased and the ability of HIV-1 Nef protein was enhanced by transfection with GSK-3β-DN in promoting vIL-6 induced microtubule formation （3.42 vs 2.51,t =3.67,P〈0.01） and angiogenesis （6.25 vs 3.97,t=4.06,P〈0.01）.In contrast,the activity of GSK-3β was significantly increased and these functions of HIV-1 Nef protein mentioned above were inhibited by transfection with GSK-3β-S9A （0.62 vs2.51,t=8.48,P〈0.01; 0.39 vs 3.97,t=8.59,P〈0.01）.The results of Western blot showed that with the elevated level of,β-catenin （in cells：3.53 vs 2.07,t=6.60,P〈0.05; in tissues：2.76 vs 1.74,t=17.40,P〈0.01） and vascular endothelial growth factor （VEGF,in cells：2.68 vs 1.87,t=4.28,P〈0.01; in tissues：2.20 vs 1.39,t=7.08,P〈0.01） were increased in the GSK-3β-DN transfected cells or tissues,while the opposite results were achieved in the GSK-3β-S9A-transfected cells （GSK-3β phosphorylation：0.50 vs 1.47,t=7.33,P〈0.01; β-catenin：1.05 vs 2.62,t=29.50,P〈0.01; VEGF：0.74 vs 2.16,t=20.95,P〈0.01） or tissues （GSK-3β phosphorylation：0.35 vs 1.97,t=10.72,P〈0.01; β-caten

关 键 词：人免疫缺陷病毒1型 负调控因子 糖原合成酶激酶3 β连环素 疱疹病毒8型 人 白细胞介素6 血管生成 

分 类 号：R512.91[医药卫生—内科学]