利用Pdl1敲除的巨噬细胞转录谱分析结核感染中PD-L1作用相关的候选基因

Screening of PD-L1 related specific candidate genes in tuberculosis by transcriptome sequencing and analysis in mycobacterium tuberculosis infected Pdl1 knockout macrophages

目的分析结核菌(Mtb)感染的程序性死亡配体1(Pdl1)特异性敲除的巨噬细胞(MΦ)转录谱变化,筛选MΦ内PD-L1作用相关的候选基因。方法构建敲除小鼠(Pdl1^Flox/-),通过Cre-loxp技术培育出MΦ上Pdl1特异性敲除的纯合和杂合小鼠(Pdl1^Flox/Flox-Cre,Pdl1^Flox/--Cre),用PCR和流式细胞术验证。分离上述小鼠腹腔MΦ,利用转录组测序技术检测Mtb感染MΦ24 h的表达谱,以同窝阴性小鼠(Pdl1^Flox/Flox)为对照,进行生物信息学分析。结果PCR和流式细胞术证实小鼠敲除成功。纯合、杂合及同窝阴性小鼠感染前后相比,差异表达基因最显著富集项为参与免疫反应和免疫过程的GO(P<0.01;P<0.01),筛出17个PD-L1相关的候选基因。通过KEGG富集分析,最显著富集的通路有Toll样受体、NF-κB信号通路等(P<0.01;P<0.01),筛出6个候选基因。结论通过转录谱分析,确定与免疫反应及免疫过程有关的Ccl2、Qa5、Il12a等17个基因,免疫及炎症相关代谢通路的Ptgs2、Cd40、Card11等6个基因为Mtb感染的MΦ内PD-L1相关的候选基因,除Il6外均为本研究新筛选的候选基因。

Objective To analyze the transcription profile of Mycobacterium tuberculosis(Mtb)-infected macrophages in which the Pdl1 gene has been knocked out and screen PD-L1 related candidate genes in Mtb-infected macrophages(MΦ).Methods Pdl1Flox/-mice were constructed,and homozygous(Pdl1^Flox/Floxwith Cre)and heterozygous(Pdl1^Flox/-with Cre)mice with targeted inactivation of the Pdl1 gene in MΦs were bred by the Cre/loxP technique and verified by PCR and FACS.Peritoneal MΦs were isolated from different substrains of mice,and those isolated from littermate negative mice(Pdl1^Flox/Flox)were used as controls.Twenty-four hours after infection,the transcript profiles in the infected and uninfected groups were sequenced by RNA-Seq,and bioinformatics analysis was performed.Results Pdl1-specific knockout mice were successfully generated and identified by PCR and FACS.The Pdl1^Flox/Flox with Cre,Pdl1^Flox/-with Cre,and Pdl1Flox/Flox infected groups were compared with their corresponding non-infected groups.Gene Ontology(GO)functional enrichment analysis showed that the most significant enrichments were immune response(P<0.01)and immune system processes(P<0.01).The seventeen candidate genes identified were regarded as specific genes related to PD-L1 in Mtb infection.Through the Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis,the most significantly enriched pathways included Toll-like receptor(P<0.01)and nuclear factor kappa-B(NF-κB)(P<0.01)signaling pathways,and six candidate genes related to PD-L1 were screened.Conclusions Through transcription analysis,seventeen genes related to the immune response,including Ccl2,Qa5,and Il12a,and six genes in immune-and inflammation-related metabolic pathways,including Ptgs2,Cd40,and Card11,were identified as specific candidate genes related to PD-L1 in macrophages infected with Mtb.Except for Il6,all candidate genes were selected for this research.