PTPRZ1高表达胶质瘤干细胞诱导TAM向M2免疫抑制表型极化的机制研究  

Mechanism of glioma stem cells with high expression of PTPRZ1 inducing TAMs polarization to M2 immunosuppressive phenotype

作　　者：安乐乐 杨莹 刘庆 窦飞越 王路静 程玥 王超 阮潜瑛 周磊 郭海涛 孔维凯 李学刚 兰川 李飞 时雨 AN Lele;YANG Ying;LIU Qing;DOU Feiyue;WANG Lujing;CHENG Yue;WANG Chao;RUAN Qianying;ZHOU Lei;GUO Haitao;KONG Weikai;LI Xuegang;LAN Chuan;LI Fei;SHI Yu(Department of Pathology,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038;Yu-Yue Pathology Scientific Research Center,Jinfeng Laboratory,Chongqing,400039,China;Department of Blood Transfusion,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038;Department of Neurosurgery,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038)

机构地区：[1]陆军军医大学(第三军医大学)第一附属医院病理科,重庆400038 [2]金凤实验室,渝粤病理科学研究中心,重庆400039 [3]陆军军医大学(第三军医大学)第一附属医院输血科,重庆400038 [4]陆军军医大学(第三军医大学)第一附属医院神经外科,重庆400038

出　　处：《陆军军医大学学报》2024年第8期796-803,共8页Journal of Army Medical University

基　　金：国家重点研发计划干细胞专项青年科学家项目(2021YFA1103000);国家自然科学基金面上项目(82373191);国家自然科学基金青年基金项目(82102889);重庆市自然科学基金博士后科学基金项目(cstc2021jcyj-bshX0063)。

摘　　要：目的探究蛋白酪氨酸磷酸酶受体Z1型(protein tyrosine phosphatase receptor-type Z1,PTPRZ1)高表达胶质瘤干细胞(glioma stem cells,GSCs)对肿瘤相关巨噬细胞(tumor-associated macrophage,TAM)表型极化和吞噬能力的影响及其调控机制。方法利用流式细胞术分选人胶质母细胞瘤(glioblastoma,GBM)样本中GSCs和非干性肿瘤细胞(non-stem tumor cells,NSTCs),并检测PTPRZ1表达。用胶质瘤细胞条件培养基或趋化因子配体20(chemokine C-C motif ligand 20,CCL20)诱导外周单核细胞(peripheral blood mononuclear cells,PBMC)来源的CD14阳性单核细胞向TAM极化。通过CRISPR/Cas9技术构建PTPRZ1稳定敲除的GSCs细胞株(PTPRZ1-KO GSCs)。利用流式细胞术检测TAM吞噬GSCs、NSTCs、PTPRZ1-Control GSCs(PTPRZ1-Ctrl GSCs)和PTPRZ1-KO GSCs的能力和TAM免疫抑制(M2)表型极化标记物CD163的表达。于基因表达综合数据库(gene expression omnibus,GEO)中配对GSCs和NSTCs转录组测序数据(GSE54791)中获得差异表达基因;于癌症和肿瘤基因图谱(the cancer genome atlas,TCGA)数据库GBM队列中鉴定预后不良基因集。上述基因集与编码人膜蛋白基因集取交集获得PTRRZ1,利用基因集富集分析(gene set enrichment Analysis,GSEA)比较TCGA数据库中高表达和低表达PTPRZ1的GBM样本的差异通路。利用转录组测序鉴定PTPRZ1-Ctrl GSCs和PTPRZ1-KO GSCs组中差异表达基因并通过RT-qPCR和Western blot验证。结果与NSTCs比较,GSCs抵抗TAM吞噬的能力较强(P<0.05)且特异性高表达PTPRZ1。PTPRZ1-KO GSCs抵抗TAM吞噬的能力显著下降(P<0.01)。高表达PTPRZ1的GBM样本中吞噬相关通路活化显著受抑(P<0.05)。PTPRZ1-KO GSCs显著下调M2型TAM极化相关趋化因子CCL20的表达(P<0.05)。CCL20重组蛋白诱导M2型TAM标记物CD163表达升高。结论PTPRZ1高表达GSCs介导TAM向M2型极化并抑制其吞噬能力,机制可能与PTPRZ1高表达GSCs上调CCL20表达有关。Objective To explore the effect of glioma stem cells with high expression of protein tyrosin phosphatase receptor type Z1(PTPRZ1)on the phenotypic polarization and phagocytosis of tumor-associated macrophages and its regulatory mechanism.Methods GSCs and non-stem tumor cells(NSTCs)were screened out from human glioblastoma(GBM)specimens using flow cytometry,and the PTPRZ1 expression in paired GSCs and NSTCs were detected.Human peripheral blood mononuclear cells(PBMC)-derived CD14+monocytes were exposed to the conditioned medium from glioma cells or recombinant chemokine C-C motif ligand 20(CCL20)for TAM polarization.Stable PTPRZ1 knockout GSCs(PTPRZ1-KO GSCs)were constructed using CRISPR/Cas9.TAM phagocytosis to GSCs,NSTCs,PTPRZ1-Control GSCs(PTPRZ1-Ctrl GSCs)and PTPRZ1-KO GSCs and the expression of immunosuppressive phenotype(M2)polarization marker CD163 were examined using flow cytometry.Differentially expressed genes(DEGs)between paired GSCs and NSTCs were determined using a bulk RNA-sequencing dataset(GSE54791)from Gene Expression Omnibus(GEO).A gene set informing worse outcome of patients with GBM was generated using The Cancer Genome Atlas(TCGA)-GBM cohort.By intersecting the aforementioned gene set with the gene set that encodes for human membrance proteins,the PTPRZ1 gene is obtained.Gene set enrichment analysis(GSEA)was used for pathway enrichment analysis to compare the differentially regulated pathways between GBMs with high or low PTPRZ1 expression.Bulk RNA sequencing,qRT-PCR and Western blotting were used to identify the DEGs between PTPRZ1-KO GSCs and PTPRZ1-Ctrl GSCs.Results GSCs were more capable of escaping from TAM phagocytosis than NSTCs(P<0.05)and had specifically up-regulated PTPRZ1 expression.PTPRZ1-KO significantly suppressed GSCs escaping from TAM phagocytosis(P<0.01).GBMs with high PTPRZ1 expression showed significant inhibition of pathways mediating phagocytosis(P<0.05).The expression of CCL20 as a M2 TAM polarization chemokine was significantly down-regulated in PTPRZ1-KO GSCs(P<0.05).Tr

关 键 词：胶质瘤干细胞 肿瘤相关巨噬细胞 细胞吞噬 PTPRZ1 

分 类 号：R394.3[医药卫生—医学遗传学] R730.23[医药卫生—基础医学] R730.264