胰腺肿瘤细胞中音猬因子调控诱导巨噬细胞极化的机制研究  

Pancreatic tumor cell induction of macrophage polarization through sonic hedgehog regulation of interferon regulatory factors

作　　者：黄友泽 陈以勒 姜跃炜 赵春[1] HUANG Youze;CHEN Yile;JIANG Yuewei;ZHAO Chun(Wenzhou TCM Hospital of Zhejiang Chinese Medical University,Wenzhou 325000,Zhejiang,China)

机构地区：[1]浙江中医药大学附属温州市中医院,浙江省温州325000

出　　处：《现代实用医学》2024年第4期433-437,共5页Modern Practical Medicine

基　　金：温州市科技计划项目(Y20190458)。

摘　　要：目的探讨胰腺肿瘤细胞中音猬因子(Shh)调控诱导巨噬细胞极化的作用机制。方法收集胰腺肿瘤组织和癌旁组织,免疫组化检测Shh表达水平;分析TCGA数据库中Shh表达差异与胰腺肿瘤患者预后的关系;体外共培养胰腺肿瘤细胞与巨噬细胞(RAW264.7),流式细胞术鉴定巨噬细胞极化,Western blot实验检测Shh蛋白达水平,RT-qPCR实验检测Shh、一氧化氮合酶(iNOS)、精氨酸酶-1(Arg-1)mRNA相对表达量;将共培养细胞分为空白对照组、阴性对照组、Shh组和Shh-RNAi组,分别感染慢病毒载体调控Shh表达,检测上述相同项目,并采用Western blot和RT-qPCR实验分别检测干扰素调节因子(IRF)4、IRF5表达水平。结果免疫组化结果显示,胰腺癌组织Shh阳性细胞率显著高于癌旁组织(P<0.05);TCGA数据分析结果显示,胰腺癌患者Shh表达显著高于正常对照(P<0.05),Shh低表达与患者远期生存率提高相关(P<0.05);与正常RAW264.7细胞比较,胰腺肿瘤共培养RAW264.7细胞Shh蛋白表达量、Shh、iNOS、Arg-1mRNA相对表达量及巨噬细胞M2/M1比例均升高(均P<0.05);与空白对照组和阴性对照组比较,Shh组Shh、IRF4蛋白、mRNA相对表达量、Arg-1 mRNA相对表达量上调,M2/M1比率升高,IRF5蛋白、mRNA相对表达量、iNOS mRNA相对表达量下调(均P<0.05);与Shh组比较,Shh-RNAi组Shh、IRF4蛋白、mRNA相对表达量、Arg-1 mRNA相对表达量下调,M2/M1比率均降低(均P<0.05),IRF5蛋白、mRNA相对表达量、iNOS mRNA相对表达量上调(均P<0.05)。结论胰腺肿瘤细胞中Shh呈高表达,过表达Shh具有促进M2型巨噬细胞极化、抑制M1型巨噬细胞极化的作用,可能是通过调控IRF4、IRF5蛋白和基因表达实现。Objective To explore the specific role mechanism of sonic hedgehog(Shh)regulating macrophage polarization in pancreatic tumor cells.Methods Clinically isolated pancreatic tumor tissues and adjacent tissues were collected,and the expression level of Shh was detected by immunohistochemistry.The relationship between Shh expression difference in TCGA database and prognosis inpatients with pancreatic cancer was analyzed.Pancreatic tumor cells and macrophages were co-cultured in vitro.The macrophage polarization was identified by flow cytometry and the level of Shh protein was detected by Western blot.RT-qPCR assay was used to detect the mRNA relative expression levels of Shh,nitric oxide synthase(iNOS)and arginase-1(Arg-1).The co-cultured cells were divided into four groups,including control group,NC group,Shh group and Shh-RNAi group.The Shh expression was regulated by lentivirus vector,and the same items were detected.The expression levels of interferon regulatory factor(IRF)4and IRF5 were detected by Western blot and RT-qPCR respectively.Results Immunohistochemistry showed that the Shh po sitive cell rate in pancreatic cancer tissues was significantly higher than that in adj acent tissues(P<0.05).TCGA data analysis revealed that the expression of Shh in patients with pancreatic cancer was significantly higher than that in normal controls(P<0.05),and the low expression of Shh was correlated with the improvement of longterm survival rate(P<0.05).Compared with normal RAW264.7 cells,the expression of Shh protein,mRNA relative expressions of Shh,iNOS and Arg-1 and M2/M1 ratio in co-cultured RAW264.7 cells in pancreatic tumors were increased(all P<0.05).Compared with control group and negative control group,the protein and mRNA relative expre ssion levels of Shh and IRF4 and mRNA relative expression level of Arg-1 in Shh group were up-regulated,the macrophage M2/M1 ratio was increased,and the protein and mRNA relative expression levels of IRF5 and mRNA relative expre ssion level of iNOS were down-regulated(all P<0.05).Co

关 键 词：胰腺肿瘤细胞 巨噬细胞 SHH 干扰素调节因子 

分 类 号：R735.9[医药卫生—肿瘤]