Expression of inherited retinal disease related genes in human microglia
10.3760/cma.j.cn511434-20241101-00402
- VernacularTitle:遗传性视网膜变性疾病致病基因在小胶质细胞中的表达研究
- Author:
Jia XU
1
;
Sijian YU
1
;
Jinyi WANG
1
;
Zibing JIN
1
Author Information
1. 首都医科大学附属北京同仁医院眼科 北京市眼科研究所，北京 100730
- Publication Type:Journal Article
- Keywords:
Inherited retinal disease;
Microglia;
Induced pluripotent stem cell;
Cell differentiation;
Gene expression
- From:
Chinese Journal of Ocular Fundus Diseases
2025;41(3):213-220
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To observe the expression of genes related to hereditary retinal diseases (IRD) in human microglia (hMG).Methods:A experimental study. Efficient differentiation of human induced pluripotent stem cells (iPSC) into hMG. Identification of octamer-binding transcription factor 4 (OCT4), sex-determining transcription factor 2 (SOX2), Nanog homeobox (NANOG), stage-specific embryonic antigen-4 (SSEA4), alpha-fetoprotein (AFP), α-smooth muscle actin (α-SMA) as markers associated with iPSC dryness and pluripotency by immunofluorescence staining Glial fibrillary acidic protein (GFAP); hMG associated marker transmembrane protein 119 (TMEM119), purinergic receptor P2Y12 (P2RY12), and allograft inflammatory factor 1 (IBA1). The proportion of CD11b + and CD45 + cells was detected by flow cytometry. Mature hMG was collected and stimulated with lipopolysaccharide for 0, 4, 8 and 12 h, and were divided into groups 0 h, 4 h, 8 h and 12 h, respectively. Total RNA samples from the 4 groups were extracted for transcriptome sequencing, and the persistently significant differentially expressed genes (DEG) were screened. Real-time quantitative polymerase chain reaction (qPCR) was used to verify and analyze the expression of DEG mRNA. The two-tailed Student t test was used for comparison between the two groups. Results:iPSC expressed the dry related markers OCT4, SOX2, NANOG and SSEA4, and differentiated into endoderm, mesoderm and ectoderm, expressing the corresponding markers AFP, α-SMA and GFAP, respectively. iPSC formed embryoid bodies under specific culture conditions, and then differentiated into hMG, and hMG expressed related markers TMEM119, P2RY12 and IBA1 by immunofluorescence staining. The double positive ratio of CD11b + and CD45 + was > 95%. Transcriptomic analysis showed that the expression of 18 DEG in hMG stimulated by LPS was changed. qPCR test results showed that compared with group 0 h, mRNA expressions of Toll-like receptor 4 (TLR4), phosphoglycerate kinase 1, disintegrin and metallopeptidase domain 9 (ADAM9) in LPS stimulated group 4 h were significantly increased ( t=25.43, 15.54, 6.26; P<0.01). The mRNA expression levels of MER proto-oncogene tyrosine kinase (MERTK), non-hydrolase domain containing lysophospholipase 12 (ABHD12), retinal dehydrogenase 11 (RDH11), DNA damage autophagic regulator 2 (DRAM2) decreased ( t=5.94, 14.14, 8.21, 6.97; P<0.01), and the differences were statistically significant. Compared with group 0 h, mRNA expressions of RDH11, MERTK, ABHD12, DRAM2 and ADAM9 in group 8 h stimulated by LPS were significantly decreased, with statistical significance ( t=25.97, 5.47, 43.97, 38.40, 3.84; P<0.05). Compared with the group 0 h, the mRNA expressions of TLR4, ADAM9, MERTK, ABHD12, RDH11 and DRAM2 in the 12 h stimulated group were significantly decreased, and the differences were statistically significant ( t=6.39, 46.11, 5.34, 14.14, 25.97, 25.65; P<0.05). Conclusion:IRD-related genes may be involved in the occurrence and development of IRD by regulating the function of hMG.
