RB1缺乏导致视网膜母细胞瘤(Rb),眼内最常见的恶性肿瘤。肿瘤相关巨噬细胞(TAMs)与局部炎症相关,特别是通过增加细胞因子和免疫逃逸。小胶质细胞,独特的视网膜稳态的常驻巨噬细胞,是Rb最重要的免疫细胞。然而,RB1缺乏是否影响小胶质细胞功能尚不清楚.在这项研究中,从Rb患者来源的人诱导多能干细胞(hiPSCs)和人胚胎干细胞(hESCs)成功分化出小胶质细胞,然后我们通过实时成像吞噬实验研究了RB1在小胶质细胞中的功能,免疫荧光,RNA-seq,qRT-PCR,ELISA和视网膜类器官/小胶质细胞共培养。RB1在小胶质细胞中大量表达,主要位于细胞核中。然后,我们在体外检查了iMGs的吞噬能力和分泌功能。我们发现,通过活成像,RB1缺乏不会影响小胶质细胞特异性标志物的表达或这些细胞的吞噬能力。LPS刺激后,缺乏RB1的小胶质细胞显示增强的先天免疫反应,MAPK信号通路激活和IL-6和TNF-α在mRNA和蛋白质水平的表达升高证明,与野生型小胶质细胞相比。此外,当视网膜类器官与缺乏RB1的小胶质细胞共培养时,观察到视网膜结构破坏,强调小胶质细胞对Rb发育的潜在贡献和视网膜母细胞瘤的潜在治疗策略。
RB1 deficiency leads to retinoblastoma (Rb), the most prevalent intraocular malignancy. Tumor-associated macrophages (TAMs) are related to local inflammation disorder, particularly by increasing cytokines and immune escape. Microglia, the unique resident macrophages for retinal homeostasis, are the most important immune cells of Rb. However, whether
RB1 deficiency affects microglial function remain unknown. In this study, microglia were successfully differentiated from Rb patient- derived human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), and then we investigated the function of
RB1 in microglia by live imaging phagocytosis assay, immunofluorescence, RNA-seq, qRT-PCR, ELISA and retina organoids/microglia co-culturing.
RB1 was abundantly expressed in microglia and predominantly located in the nucleus. We then examined the phagocytosis ability and secretion function of iMGs in vitro. We found that RB1 deficiency did not affect the expression of microglia-specific markers or the phagocytic abilities of these cells by live-imaging. Upon LPS stimulation,
RB1-deficient microglia displayed enhanced innate immune responses, as evidenced by activated MAPK signaling pathway and elevated expression of IL-6 and TNF-α at both mRNA and protein levels, compared to wildtype microglia. Furthermore, retinal structure disruption was observed when retinal organoids were co-cultured with RB1-deficient microglia, highlighting the potential contribution of microglia to Rb development and potential therapeutic strategies for retinoblastoma.