Chinese 1型弓形虫眼病小鼠NK细胞亚群的初步分析

Preliminary analysis of NK cell subsets in murine model of ocular toxoplasmosis with Chinese 1 strain

目的建立我国Chinese 1优势基因型获得性弓形虫眼病小鼠模型,分析弓形虫感染对眼组织局部NK细胞及CD49a^(+)NK细胞亚群的影响。方法C57BL/6小鼠经口感染20个弓形虫Wh6包囊,30d后取眼组织,苏木精和伊红(H&E)染色检查病理变化;采用PCR扩增眼组织中的弓形虫ITS-1基因;采用实时荧光定量PCR(qRT-PCR)和免疫组化方法分析眼组织中的干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)等细胞因子的mRNA及蛋白表达水平;对眼组织转录组测序,进行基因表达差异分析、GO(gene ontology)富集分析和KEGG(Kyoto Encyclopedia of Genes and Genomes)富集分析;采用流式细胞术检测眼组织中NK细胞和CD49a^(+)NK细胞亚群水平。结果H&E染色检查感染组小鼠视网膜发生严重的病理改变,PCR扩增眼组织中弓形虫特异性基因ITS-1阳性,qRT-PCR扩增眼组织中细胞因子IFN-γ、TNF-α的mRNA表达上调(P<0.01),免疫组化检查视网膜中IFN-γ、TNF-α表达上调。转录组测序显示,感染组小鼠眼组织中有601个基因表达上调,108个基因表达下调,GO富集分析中差异表达基因在免疫反应、炎症反应等生物过程显著富集,KEGG富集分析显示31个差异表达基因富集在NK细胞介导的细胞毒性通路。流式细胞术检测感染鼠眼组织中NK和CD49a^(+)NK细胞百分比显著增加(P<0.01)。结论成功构建了Chinese 1优势基因型获得性弓形虫眼病小鼠模型,感染鼠眼组织中NK细胞及CD49a^(+)NK细胞亚群百分比增加,提示NK细胞及CD49a^(+)NK细胞亚群在眼组织中积累可能参与弓形虫眼病的发生过程。

Objective To establish murine model of acquired ocular toxoplasmosis with the predominant clonal lineage Chinese 1 strain,and analyze the effects of Toxoplasma gondii infection on theproportion of NK cells and CD49 a^(+) NK subsets in oculartissue.Methods C57 BL/6(B6) mice were infected perorally with 20 cysts of the TgCtWh6 strain,and the ocular tissuewere isolatedat 30 days after infection.Hematoxylin and eosin(H&E) staining was used to detect ocular pathological changes.The expression of Toxoplasmagondii-specificgene ITS-1 was detected by PCR amplification.The expression of interferon-gamma(IFN-γ) and tumor necrosis factor-α(TNF-α) were analyzed by quantitative real-time polymerase chain reaction(qRT-PCR) and immunohistochemical staining.The differentially expressed genes in ocular tissues were detected by RNA sequencing,and then GO enrichment and KEGG enrichment were analyzed.The percentage of NK cells and CD49 a^(+) NK cells in oculartissue was analyzed by flow cytometry.Results The results showed that infected mice had severe ocular damage and PCR amplification of Toxoplasmagondii-specificgene ITS-1 were positive in infected ocular tissue.The mRNA expression of cytokines IFN-γ and TNF-α was upregulated in infected ocular tissue by qRT-PCR amplification(P<0.01),and the protein expression was upregulated in infected retina by immunohistochemical analysis.RNA sequencing showed that 601 genes were up-regulated and 108 genes were down-regulated in infected ocular tissues.In GO enrichment analysis,the differentially expressed genes were significantly enriched in immune response and inflammatory responsein biological processes.KEGG enrichment analysis showed that 31 differentially expressed genes were enriched in the NK-mediated cytotoxicity pathway.The percentage of NK cells and CD49 a^(+) NK cells were significantly increased in infected ocular tissue(P<0.01).Conclusion The murine model of acquired ocular toxoplasmosis with the predominant clonal lineage Chinese 1 strain has been successfully established.The increased percentage of NK cells and CD49 a^(+) NK cells in infected ocular tissue suggests that the accumulation of NK cells and CD49 a^(+) NK subsets in ocular tissues may be involved in the process of ocular toxoplasmosis.