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  

Expression of inherited retinal disease related genes in human microglia

作　　者：许佳 俞思健 王瑾宜 金子兵 Xu Jia;Yu Sijian;Wang Jinyi;Jin Zibing(Department of Ophthalmology,Capital Medical University,Beijing Tongren Hospital,Beijing Institute of Ophthalmology,Beijing 100730,China)

机构地区：[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

出　　处：《中华眼底病杂志》2025年第3期213-220,共8页Chinese Journal of Ocular Fundus Diseases

基　　金：国家自然科学基金(82101145);国家自然科学基金杰出青年科学基金(82125007)。

摘　　要：目的观察遗传性视网膜疾病(IRD)相关基因在人小胶质细胞(hMG)中的表达情况。方法实验研究。将人诱导多能干细胞(iPSC)高效分化成hMG。采用免疫荧光染色鉴定iPSC干性和多能性相关标志物八聚体结合转录因子4(OCT4)、性别决定区转录因子2(SOX2)、Nanog同源盒(NANOG)、阶段特异性胚胎抗原-4(SSEA4)、甲胎蛋白(AFP)、α-平滑肌肌动蛋白(α-SMA)、神经胶质纤维酸性蛋白(GFAP);hMG相关标志物跨膜蛋白119(TMEM119)、嘌呤能受体P2Y12(P2RY12)和同种异体移植物炎症因子1(IBA1)。流式细胞仪检测CD11b+和CD45+双阳性的细胞比例。收集成熟的hMG,分别用脂多糖刺激0、4、8、12 h,并以此分别为0 h组、4 h组、8 h组、12 h组。随后提取4组的总RNA样本进行转录组测序,筛选持续显著差异表达基因(DEG)。采用实时荧光定量聚合酶链反应(qPCR)验证并分析DEG mRNA表达情况。两组间比较采用双尾Student t检验,结果iPSC表达干性相关标志物OCT4、SOX2、NANOG和SSEA4,并能分化为内胚层、中胚层和外胚层,分别表达相应层的标志物AFP、α-SMA和GFAP。iPSC在特定培养条件下形成拟胚体,进而分化为hMG,并成功表达hMG相关标记物TMEM119、P2RY12和IBA1。流式细胞仪检测发现,CD11b+和CD45+双阳性比例>95%。转录组学分析结果显示,在脂多糖刺激的hMG中共筛选出18个DEG.qPCR检测结果显示,与0 h组对比,脂多糖刺激4 h组Toll样受体4(TLR4)、磷酸甘油酸激酶1、解整合素和金属肽酶结构域9(ADAM9)mRNA表达量显著升高(t=25.43、15.54、6.26,P<0.01),MER原癌基因酪氨酸激酶(MERTK)、含有12的溶血磷脂酶的非水解酶结构域(ABHD12)、视黄醛脱氢酶11(RDH11)、DNA损伤自噬调节剂2(DRAM2)mRNA表达量降低(t=5.94、14.14、8.21、6.97,P<0.01),差异均有统计学意义;脂多糖刺激8 h组RDH11、MERTK、ABHD12、DRAM2、ADAM9 mRNA表达量显著下降,差异均有统计学意义(t=25.97、5.47、43.97、38.40、3.84,P<0.05);脂�ObjectiveTo observe the expression of genes related to hereditary retinal diseases(IRD)in human microglia(hMG).MethodsA 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.ResultsiPSC 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

关 键 词：遗传性视网膜疾病 小胶质细胞 人诱导多能干细胞 细胞分化 基因表达 

分 类 号：R73[医药卫生—肿瘤]