基于KEGG通路富集分析颗粒酶B-穿孔素选择性诱导胰腺炎时巨噬细胞凋亡的作用  

The role of granzyme B-perforin in selectively inducing apoptosis of macrophages in pancreatitis based on KEGG pathway enrichment analysis

作　　者：孔鸿儒 俞浩辉 陈格尔 陈咨苗[3] 戴胜杰 孙学成[4] 金约朋[1] 杨文军[1] 孙洪伟[1] KONG Hongru;YU Haohui;CHEN Geer;CHEN Zimiao;DAI Shengjie;SUN Xuecheng;JIN Yuepeng;YANG Wenjun;SUN Hongwei(Department of Hepatopancreatobiliary Surgery,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325015,China;The First School of Medicine,School of Information and Engineering,Wenzhou Medical University,Wenzhou 325035,China;Department of Endocrinology,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325015,China;Department of Gastroenterology,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325015,China)

机构地区：[1]温州医科大学附属第一医院肝胆胰外科,浙江温州325015 [2]温州医科大学第一临床医学院(信息与工程学院),浙江温州325035 [3]温州医科大学附属第一医院内分泌科,浙江温州325015 [4]温州医科大学附属第一医院消化内科,浙江温州325015

出　　处：《温州医科大学学报》2022年第12期972-980,共9页Journal of Wenzhou Medical University

基　　金：温州市基础性科研项目(Y20180077,Y20180103)。

摘　　要：目的:从分子水平探究颗粒酶B(GRB)-穿孔素(PFP)对选择性诱导胰腺炎时巨噬细胞凋亡的影响。方法:采用肿瘤坏死因子α(TNF-α)刺激人单核巨噬细胞系(TPH-1)模拟急性胰腺炎时巨噬细胞炎症反应,通过CCK8法筛选TNF-α、PFP、GRB的最适浓度;流式细胞仪分别检测对照组、TNF-α组、PFP+GRB组、TNF-α+PFP+GRB组的细胞凋亡情况。将细胞样本分为3组:正常细胞组(NC组)、TNF-α处理组(Treat1组)和TNF-α+PFP+GRB处理组(Treat2组)。提取3组细胞RNA进行转录组学分析,分别通过主成分分析、差异基因分析、富集分析检测各组基因表达情况;选取“细胞因子-细胞因子受体相互作用通路”中的7个差异表达基因(RAB37、GPA33、USH1C、SYTL1、MSRB3、GPER1和CD1B)应用荧光定量PCR验证转录组学分析的准确性。结果:Treat2组与Treat1及NC组相比,细胞凋亡比例显著升高(P<0.05);NC组与Treat1组、Treat2组的差异比较大,显著差异基因较多,而Treat1组与Treat2组的显著差异基因较少,但Treat2组比Treat1组相对于NC组的变化更大;富集分析显示差异基因主要富集于“细胞因子-细胞因子受体相互作用通路”;荧光定量PCR结果显示,所选取的7个差异基因与NC组相比明显下调(P<0.05),和转录组学结果趋势一致。结论:GRB-PFP可选择性诱导胰腺炎时巨噬细胞的凋亡,并作用于“细胞因子-细胞因子受体相互作用通路”,从而下调细胞因子表达,减轻炎症反应。Objective:To investigate the effect of granzyme B(GRB)-perforin(PFP)on macrophage apoptosis during selective induction of pancreatitis.Methods:Human mononuclear macrophage cell line(TPH-1)was stimulated by tumor necrosis factor-α(TNF-α)to simulate the macrophage microenvironment during acute pancreatitis.The optimum concentrations of TNF-α,PFP and GRB were screened by CCK8 test.Apoptosis was detected by flow cytometry in control group,TNF-αgroup,PFP+GRB group and TNF-α+PFP+GRB group.The cell samples were divided into three groups:normal cell group(NC group),TNF-αstimulation group(Treat1 group)and TNF-α+PFP+GRB stimulation group(Treat2 group).Three groups of cell RNA were extracted for transcriptomic analysis.Gene expression was detected by principal component analysis,differential gene analysis and enrichment analysis.Seven differentially expressed genes(RAB37,GPA33,USH1C,SYTL1,MSRB3,GPER1,CD1B)were selected in the“cytokine-cytokine receptor interaction pathway”to verify the accuracy of RNA-seq by fluorescence quantitative PCR.Results:Compared to NC group and Treat1 group,the apoptosis rate of Treat2 group was significantly higher.The difference between NC group and Treat1 group and Treat2 group was obvious,with more significant genes,while the difference between Treat1 group and Treat2 group was unconspicuous,but the change of Treat2 group was greater than that of Treat1 group in NC group.Enrichment analysis showed that the differential genes were mainly concentrated in the“cytokine-cytokine receptor interaction pathway”.The results of fluorescence quantitative PCR and RNA-seq were highly consistent.Conclusion:GRB-PFP selectively induces apoptosis of macrophages in pancreatitis and acts as a“cytokine-cytokine receptor interaction pathway”to down-regulate the expression of cytokines and reduces the inflammatory response.

关 键 词：胰腺炎 细胞凋亡 细胞因子 巨噬细胞 颗粒酶B 穿孔素 

分 类 号：R96[医药卫生—药理学]