HTRA3基因对脉络膜新生血管和M2型巨噬细胞极化的影响  

Effect of HtrA serine peptidase 3 gene on choroidal neovascularization and M2 macrophage polarization

作　　者：肇莉莉 王萍 孙连义 马为梅 张乐 喻磊 ZHAO Lili;WANG Ping;SUN Lianyi;MA Weimei;ZHANG Le;YU Lei(Department of Ophthalmology,Xi’an People’s Hospital(Xi’an Fourth Hospital),Shaanxi Eye Hospital,the People’s Hospital Affiliated of Northwest University,Xi’an 710004,Shaanxi Province,China;Department of Ophthalmology,Northwest Women’s and Children’s Hospital,Xi’an 710003,Shaanxi Province,China)

机构地区：[1]西安市人民医院(西安市第四医院),陕西省眼科医院,西北大学附属人民医院眼科,陕西省西安市710004 [2]西北妇女儿童医院眼科,陕西省西安市710003

出　　处：《眼科新进展》2024年第4期275-281,共7页Recent Advances in Ophthalmology

基　　金：陕西省自然科学基础研究计划项目(编号:2021JM-547);陕西省卫生健康科研基金项目(编号:2022D036)。

摘　　要：目的 探讨HtrA丝氨酸肽酶3(HTRA3)基因对脉络膜新生血管(CNV)和M2型巨噬细胞极化的影响。方法 收集30例湿性年龄相关性黄斑变性(wAMD)患者(wAMD组)和30例同期健康体检者(健康组)的空腹静脉血,通过qRT-PCR检测血清HTRA3 mRNA水平。将RF/6A细胞随机分为对照组、NC-sh组和HTRA3-sh组,使用Lipofectamine2000将NC-shRNA和HTRA3-shRNA慢病毒载体分别转染到NC-sh组和HTRA3-sh组RF/6A细胞中,通过qRT-PCR和Western blot检测HTRA3的转染情况。将RF/6A细胞随机分为N组、H组、H+NC-sh组和H+HTRA3-sh组,细胞转染后,N组RF/6A细胞在完全RPMI 1640培养基中进行常氧培养,其他组细胞在添加200 mmol·L^(-1)氯化钴(CoCl_(2))的RPMI 1640培养基中进行低氧培养,使用Matrigel测定小管形成。将C57BL/6J小鼠随机分为对照组、CNV组、CNV+NC-sh组和CNV+HTRA3-sh组,每组12只。对照组为未建模的小鼠,其他组为激光诱导的CNV模型小鼠。向CNV+NC-sh组和CNV+HTRA3-sh组小鼠玻璃体内分别注射1μL滴度为1×10^(11)TU·mL^(-1)的NC-shRNA和HTRA3-shRNA慢病毒载体。对照组和CNV组小鼠注射PBS。注射后7 d,对小鼠进行荧光素眼底血管造影(FFA)检测和眼球苏木精伊红(HE)染色。通过qRT-PCR检测RF/6A细胞或各组小鼠脉络膜组织中HTRA3、类几丁质酶3样蛋白3(Ym-1)、精氨酸酶1(Arg-1)、诱导型一氧化氮合酶(iNOS)、环氧化酶-2(COX-2)和血管内皮生长因子(VEGF)mRNA水平。通过Western blot检测RF/6A细胞或脉络膜组织中HTRA3、VEGF和细胞核核因子κB(NF-κB) p65的蛋白表达水平。结果 与健康组比较,wAMD组患者的血清HTRA3 mRNA水平升高(t=11.804,P<0.001)。与对照组和NC-sh组比较,HTRA3-sh组RF/6A细胞的HTRA3 mRNA和蛋白表达水平降低(P<0.05)。与N组比较,H组RF/6A细胞的闭合管腔数量、HTRA3和VEGF的mRNA和蛋白表达水平均增加(均为P<0.05)。与H+NC-sh组比较,H+HTRA3-sh组RF/6A细胞的闭合管腔数量、HTRA3和VEGF的mRNA和蛋白表达水平均减少(�Objective To investigate the effect of the HtrA serine peptidase 3(HTRA3)gene on choroidal neovascularization(CNV)and M2 macrophage polarization.Methods Fasting venous blood was collected from 30 patients with wet age-related macular degeneration(wAMD group)and 30 healthy subjects(normal group).The serum HTRA3 messenger ribonucleic acid(mRNA)level was detected by quantitative reverse transcription polymerase chain reaction(qRT-PCR).RF/6A cells were randomly divided into the control group,NC-sh group and HTRA3-sh group.Lentiviral vectors of NC-shRNA and HTRA3-shRNA were transfected into RF/6A cells in the NC-sh group and HTRA3-sh group by Lipofectamine2000.HTRA3 transfection was detected by qRT-PCR and Western blot.Then,the RF/6A cells were randomly divided into the N group,H group,H+NC-sh group and H+HTRA3-sh group.After cell transfection,RF/6A cells in the N group were cultured in a RPMI 1640 complete medium at a normoxia state,and cells in other groups were cultured in a RPMI 1640 medium with 200 mmol·L^(-1) CoCl_(2) at a hypoxia state.Tubule formation was measured by Matrigel.The C57BL/6J mice were divided into the control group,CNV group,CNV+NC-sh group and CNV+HTRA3-sh group,with 12 mice in each group.Mice in the control group were unmodeled mice,and mice in the other groups were laser-induced CNV model mice.NC-shRNA and HTRA3-shRNA lentiviral vectors with a titer of 1×10^(11) TU·mL^(-1) were administered to mice in the CNV+NC-sh group and CNV+HTRA3-sh group via intravitreal injection.Mice in the control group and CNV group were injected with phosphate buffered saline.After 7 days of treatment,the mice were examined by fundus fluorescein angiography,and the eyeballs received hematoxylin&eosin staining.The mRNA levels of HTRA3,chitinase-like protein 3(Ym-1),arginase 1(Arg-1),inducible nitric oxide synthase(iNOS),cyclooxygenase-2(COX-2)and vascular endothelial growth factor(VEGF)in RF/6A cells or choroidal tissues were detected by qRT-PCR.The protein expression levels of HTRA3,VEGF and nuclear factor kappa

关 键 词：湿性年龄相关性黄斑变性 脉络膜新生血管 HtrA丝氨酸肽酶3 M2型巨噬细胞极化 

分 类 号：R774.5[医药卫生—眼科]