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( VSIG4)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) VSIG4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-α mRNA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 VSIG4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。
ObjectiveTo investigate the effect and mechanism of the V-set and immunoglobulin domain-containing 4 ( VSIG4 ) gene mutation on the function of microglia in retinitis pigmentosa (RP).
MethodsLocalization of VSIG4 in the retina was detected by immunofluorescence.HMC3 cells (human microglial cells) were transfected with wild-type (Len-WT) VSIG4 gene, mutant type (Len-Mut) VSIG4 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.
ResultsVSIG4 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-1β and TNF-α mRNA 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).
ConclusionsVSIG4 is localized to mouse retinal microglia.When the VSIG4 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.
徐春龙,张国伟,杜君,等. 视网膜色素变性中 VSIG4基因变异对小胶质细胞功能的影响及其作用机制 [J]. 中华实验眼科杂志,2024,42(10)：898-908.
DOI:10.3760/cma.j.cn115989-20240304-00063版权归中华医学会所有。
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徐春龙：实验操作、资料分析、撰写及修改文章；张国伟：参与选题、设计实验；杜君：指导论文写作及修改文章；贾珍：收集及分析数据；王静萍：收集数据；王梓文：分析数据；李杨：解释数据；陆宏：参与选题、指导论文写作、对文章知识性内容的审阅及定稿
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