G蛋白抑制性α亚单位1/3介导神经轴突导向因子-1信号转导并调控血管生成  

G protein alpha inhibitory subunit 1/3 mediate Netrin-1-activated signaling and angiogenesis

作　　者：姚雨佳 李佳骏 王苏豫 李柯然 Yao Yujia;Li Jiajun;Wang Suyu;Li Keran(Department of Ophthalmology,The Affiliated Eye Hospital of Nanjing Medical University,Nanjing 210029,China)

机构地区：[1]南京医科大学眼科医院,南京210029

出　　处：《中华眼底病杂志》2024年第10期781-789,共9页Chinese Journal of Ocular Fundus Diseases

基　　金：南京市卫生科技发展专项资金项目(YKK23264);朗视界·沐光明-中青年眼科科研项目(BCF-KH-YK-20230803-07);“333人才”培养支持资助项目;江苏省研究生科研与实践创新计划(JX10414151、JX10414152)。

摘　　要：目的观察G蛋白抑制性α亚单位(Gαi)1和Gαi3对神经轴突导向因子-1(NTN1)诱导的信号转导和血管生成的影响, 探讨其可能机制。方法采用随机数字表法将20只6~8周龄雄性C57BL/6J小鼠随机分为正常对照组、糖尿病组, 每组各10只。糖尿病组通过腹腔注射链脲佐菌素建立糖尿病小鼠模型。造模12周后采用实时定量聚合酶链反应、蛋白质免疫印迹法检测各组小鼠视网膜中Ntn1、Gαi1、Gαi3的mRNA和蛋白相对表达量。采用随机数字表法将35只2周龄雄性C57BL/6J小鼠随机分为正常对照组、玻璃体腔注射NTN1组(NTN1组)、视网膜内皮细胞Gαi1/Gαi3特异性敲低+玻璃体腔注射NTN1组(Gαi1/Gαi3 eKD+NTN1组), 其中正常对照组、NTN1组每组各15只, Gαi1/Gαi3 eKD+NTN1组5只。采用同工凝集素B4染色法观察各组小鼠视网膜新生血管形成情况。将体外培养的人脐静脉内皮细胞(HUVEC)分为阴性对照慢病毒组(shC组)、阴性对照慢病毒+NTN1处理组(shC+NTN1组)、Gαi1/Gαi3敲低组(shGαi1/Gαi3组)、Gαi1/Gαi3敲低+NTN1处理组(shGαi1/Gαi3+NTN1组)。采用EdU染色检测NTN1、Gαi1、Gαi3对HUVEC增殖的影响;Transwell实验测定NTN1、Gαi1、Gαi3对HUVEC迁移能力的影响;Matrigel实验检测NTN1、Gαi1、Gαi3对HUVEC管腔形成能力的影响。采用蛋白质免疫印迹法检测HUVEC中蛋白激酶B(Akt)、磷酸化Akt(p-Akt)、核糖体蛋白S6激酶(S6K)、磷酸化S6K(p-S6K)、细胞外调节蛋白激酶(Erk1/2)、磷酸化Erk1/2(p-Erk1/2)蛋白表达量。两组间比较采用t检验;三组间比较采用单因素方差分析;多因素比较采用事后Dunnett检验。结果与正常对照组比较, 糖尿病组小鼠视网膜组织中Ntn1、Gαi1、Gαi3 mRNA和蛋白相对表达量显著增加, 差异有统计学意义(t=11.800、9.298、10.620、7.503、3.432、8.037, P<0.000 1)。与shC组比较, shGαi1/Gαi3组HUVEC中Gαi1、Gαi3 mRNA和蛋白相对表达量显著降低, 差异有统计学意义(t=16Objective To observe the effect of G protein alpha inhibitory subunit(Gai)1 and Gai3 on signal transduction and angiogenesis induced by Netrin-1(NTN1)and explore the possible mechanisms.MethodsTwenty male C57BL/6J mice aged 6 to 8 weeks were randomly assigned to a control group and a diabetic group,with 10 mice in each group.Diabete group mice were induced by streptozotocin to establish diabetes model.12 weeks after modeling,quantitative real-time polymerase chain reaction and Western blot were performed to detect the expression of Ntnl,Gail and Gai3 in diabetic retinas.Additionally,35 male C57BL/6J mice aged 2 weeks were randomly stratified into three groups:a control group,an intravitreal injection of NTN1 group(NTN1 group),and a retinal endothelial cell-specific Gail/Gai3 knockdown coupled with intravitreal NTN1 injection group(Gail/Gai3 eKD+NTN1 group),with 15 mice in each of the normal control and NTN1 groups,and 5 mice in the Gail/Gai3 eKD+NTN1 group.Isolectin B4 staining was performed to observe retinal neovascularization.In vitro,human umbilical vein endothelial cells(HUVEC)were divided into four groups:negative control lentiviral transfection group(shC group),negative control lentiviral transfection+NTN1 treatment group(shC+NTN1 group),Gail/Gai3 knockdown group(shGail/Gai3 group),and Gail/Gai3 knockdown+NTN1 treatment group(shGail/Gai3+NTN1 group).The effects of NTN1,Gail,and Gai3 on HUVEC proliferation were assessed using the EdU assay.Transwell assays were conducted to determine the effects on HUVEC migration,and Matrigel assays were used to evaluate the effects on HUVEC tube formation.Protein kinase B(Akt),phosphorylated Akt(p-Akt),ribosomal protein S6 kinase(S6K),phosphorylated S6K(p-S6K),extracellular regulatory protein kinase(Erk1/2),phosphorylated Erk1/2(p-Erk1/2)protein expression on HUVEC were detected by Western blot.Results Compared with the control group,the relative expression levels of Ntnl,Gail,and Gai3 mRNA and protein in the diabetic group retina were significantly increased,with statist

关 键 词：G蛋白 神经轴突导向因子-1 糖尿病视网膜病变 视网膜新生血管 

分 类 号：R587.2[医药卫生—内分泌] R774.1[医药卫生—内科学]