摘要
目的研究视网膜色素变性(RP)中的V-set和免疫球蛋白结构域4(VSIG 4)基因变异对小胶质细胞功能的影响及其作用机制。方法利用免疫荧光染色检测VSIG4在小鼠视网膜中的定位。给HMC3细胞(人小胶质细胞系)转染野生型(Len-WT)、突变型(Len-Mut)VSIG 4基因以及转染空载病毒(Len-Cont),并通过有或无脂多糖(LPS)刺激HMC3细胞分为对照组、LPS-Len-WT组、LPS-Len-Mut组、LPS-Len-Cont组、Len-WT组、Len-Mut组和Len-Cont组。采用实时荧光定量PCR检测炎症因子白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)的mRNA表达水平;采用Western blot法检测核因子红细胞系2相关因子2(Nrf2)、血红素加氧酶1(HO-1)、谷胱甘肽过氧化物酶4(GPX4)、核转录因子κB(NF-κB)p65亚单位(P65)和磷酸化P65(PP65)的蛋白表达水平;采用吞噬实验检测细胞吞噬功能;采用细胞划痕和Transwell实验检测细胞迁移能力。将LPS刺激状态下的HMC3细胞与661W细胞(小鼠视网膜感光细胞)共培养,采用Western blot法检测B细胞淋巴瘤因子2(Bcl-2)和Bcl-2相关X蛋白(Bax)表达水平,采用凋亡实验检测661W细胞的凋亡数量。结果VSIG4定位于小鼠视网膜小胶质细胞。实时荧光定量PCR检测结果显示,与LPS-Len-WT组相比,LPS-Len-Mut组HMC3细胞IL-1β、TNF-αmRNA相对表达量均显著升高,差异均有统计学意义(均P<0.05)。Western blot检测结果显示,与LPS-Len-WT组相比,LPS-Len-Mut组HMC3细胞Nrf2、HO-1、GPX4的蛋白表达水平均显著降低,PP65/P65比值显著升高,差异均有统计学意义(均P<0.05)。吞噬实验检测结果显示,Len-Cont组、LPS-Len-Cont组、LPS-Len-WT组和LPS-Len-Mut组细胞吞噬率分别为(35.67±3.22)%、(63.67±10.07)%、(84.00±3.46)%和(64.67±2.31)%,各组HMC3细胞吞噬率总体比较差异有统计学意义(F=59.06,P<0.001)。与LPS-Len-WT组相比,LPS-Len-Mut组HMC3细胞吞噬率显著降低,差异有统计学意义(P<0.05)。细胞划痕和Transwell实验检测结果显示,与LPS-Len-WT组相比,LPS-Len-Mut组HMC3细胞24、48 h时迁移率和24 h时单位面积侵袭细胞数显著降低,差异均有统计学意义(均P<0.05)。与LPS-Len-WT组相比,共培养系统中LPS-Len-Mut组Bax/Bcl-2蛋白表达量比值及细胞凋亡数量显著升高,差异均有统计学意义(均P<0.05)。结论VSIG4定位于小鼠视网膜小胶质细胞。RP中的VSIG 4基因变异后,LPS刺激状态下的HMC3细胞NF-κB信号通路激活,Nrf2/HO-1信号通路活化程度减弱,细胞分泌炎症因子增多;吞噬和迁移能力减弱;共培养系统中细胞凋亡增加。
Objective To investigate the effect and mechanism of the V-set and immunoglobulin domain-containing 4(VSIG 4)gene mutation on the function of microglia in retinitis pigmentosa(RP).Methods Localization of VSIG4 in the retina was detected by immunofluorescence.HMC3 cells(human microglial cells)were transfected with wild-type(Len-WT)VSIG 4 gene,mutant type(Len-Mut)VSIG 4 gene and empty vector virus(Len-Cont)and stimulated by the presence or absence of lipopolysaccharide(LPS),then divided into control group,LPS-Len-Mut group,LPS-Len-WT group,LPS-Len-Cont group,Len-Mut group,Len-WT group and Len-Cont group.The mRNA expression levels of the inflammatory factors interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)were detected by real-time fluorescence quantitative PCR.Protein expression levels of nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase 1(HO-1),glutathione peroxidase 4(GPX4),nuclear transcription factor-κB(NF-κB)p65 subunit(P65),and phosphorylated P65(PP65)were detected by Western blot.Cell phagocytic function was detected by phagocytosis assay.Cell migration ability was detected by cell scratch and transwell migration assay.LPS-stimulated HMC3 cells were co-cultured with 661W cells(mouse retinal photoreceptor cells),and the expression levels of B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X(Bax)proteins of the cells were detected by Western blot.The number of apoptotic cells was determined by apoptosis assay.Results VSIG4 was localized to microglia in mouse retina.The real-time fluorescence quantitative PCR results showed that compared with LPS-Len-WT group,the relative expression levels of IL-1βand TNF-αmRNA in HMC3 cells were significantly increased in LPS-Len-Mut group(both at P<0.05).The Western blot results showed that compared with LPS-Len-WT group,the protein expression levels of Nrf2,HO-1,and GPX4 in HMC3 cells were significantly reduced in LPS-Len-Mut group,and the PP65/P65 ratio was significantly increased(all at P<0.05).The phagocytic experiment results showed that the phagocytic rates of HMC3 cells in Len-Cont group,LPS-Len-Cont group,LPS-Len-WT group,and LPS-Len-Mut group were(35.67±3.22)%,(63.67±10.07)%,(84.00±3.46)%,and(64.67±2.31)%,respectively,showing a statistically significant difference(F=59.06,P<0.001).Compared with LPS-Len-WT group,the phagocytic rate of HMC3 cells was significantly reduced in LPS-Len-Mut group(P<0.05).The results of cell scratch and transwell migration assay showed that compared with LPS-Len-WT group,the migration rate of HMC3 cells at 24 and 48 hours and the number of invading cells per unit area at 24 hours were significantly reduced in LPS-Len-Mut group(all at P<0.05).Compared with LPS-Len-WT group,the expression ratio of Bax/Bcl-2 protein and the number of cell apoptosis were significantly increased in the LPS-Len-Mut group under the co-culture system(both at P<0.05).Conclusions VSIG4 is localized to mouse retinal microglia.When the VSIG 4 gene in RP mutates,HMC3 cells under LPS stimulation exhibit a series of changes,including activation of the NF-κB signaling pathway,decreased activation of the Nrf2/HO-1 signaling pathway,increased secretion of inflammatory cytokines,reduced phagocytic and migratory abilities,and increased cell apoptosis in co-culture systems.
作者
徐春龙
张国伟
杜君
贾珍
王静萍
王梓文
李杨
陆宏
Xu Chunlong;Zhang Guowei;Du Jun;Jia Zhen;Wang Jingping;Wang Ziwen;Li Yang;Lu Hong(Department of Ophthalmology,Affiliated Hospital of Nantong University,Medical School of Nantong University,Nantong 226001,China)
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2024年第10期898-908,共11页
Chinese Journal Of Experimental Ophthalmology
基金
江苏省自然科学基金(BK20221273)。
