PI3Kγ调控肿瘤相关巨噬细胞表型影响胆管癌细胞EMT进程与干性特征  

PI3Kγregulates tumor-associated macrophage phenotype and influences EMT progression and stem characteristics of cholangiocarcinoma cells

作　　者：唐津天[1] 唐润娟[2] 薛峰[1] 黎旺红 TANG Jintian;TANG Runjuan;XUE Feng;LI Wanghong(The Second Ward of Hepatobiliary Pancreatic Surgery,Affiliated Tumor Hospital of Xinjiang Medical University,Urumqi 830000;Department of Rehabilitation,the Second Affiliated Hospital of Xinjiang Medical University,China)

机构地区：[1]新疆医科大学附属肿瘤医院肝胆胰外科二病区,新疆乌鲁木齐830000 [2]新疆医科大学第二附属医院康复科

出　　处：《胃肠病学和肝病学杂志》2024年第2期190-196,共7页Chinese Journal of Gastroenterology and Hepatology

基　　金：新疆维吾尔自治区自然科学基金资助项目(2021D01C399)。

摘　　要：目的探究磷酸肌醇3-激酶γ(phosphatidylinositide 3-kinaseγ,PI3Kγ)调控肿瘤相关巨噬细胞(tumor-associated macrophage,TAM)表型影响胆管癌细胞EMT进程及干性特征的作用。方法利用佛波酯(Phorbol 12-myristate 13-acetate,PMA)和IL-4诱导建立M2型TAM,并使用PI3Kγ抑制剂AS605240进行干预,观察细胞形态变化,免疫细胞荧光染色检测细胞表面M2特异性表型标志物CD163表达情况。建立M2型TAM与人胆管癌细胞系QBC-939共培养体系,分组为对照组、M2组、PI3Kγ抑制剂组,进行对应处理后收集QBC-939细胞,Transwell实验检测细胞迁移与侵袭能力,免疫细胞荧光染色观察细胞内E-cadherin与Vimentin表达,Western blotting检测细胞中E-cadherin、N-cadherin、Vimentin、MMP2及MMP9蛋白表达,肿瘤细胞成球实验检测细胞干性,Western blotting检测细胞中肿瘤干细胞相关蛋白CD133、OCT4及SOX2蛋白表达。结果经PMA和IL-4诱导后,细胞呈典型M2型形态,CD163荧光强度明显增加,但经PI3Kγ抑制剂干预后,M2型细胞数目明显减少,CD163荧光强度减弱。与M2组比较,PI3Kγ抑制剂组细胞迁移数目与侵袭数目均减少(P<0.05),E-cadherin荧光密度值升高、Vimentin荧光密度值降低(P<0.05),E-cadherin蛋白表达水平上调而N-cadherin、Vimentin、MMP2、MMP9蛋白表达水平均下调(P<0.05),同时,肿瘤细胞成球数量减少(P<0.05),CD133、OCT4及SOX2蛋白表达水平均下调(P<0.05)。结论抑制PI3Kγ能够抑制M2型TAM表型转化,从而抑制胆管癌细胞的EMT进程及肿瘤干性特征,发挥抗癌作用。Objective To explore the effect of phosphatidylinositide 3-kinaseγ(PI3Kγ)on the EMT process and dry characteristics of cholangiocarcinoma cells by regulating tumor-associated macrophage(TAM)phenotype.[WTHZ]Methods Phorbol 12-myristate 13-acetate(PMA)and IL-4 were used to induce the establishment of M2-type TAM,and at the same time,PI3Kγinhibitor AS605240 was used to intervene.The cell morphology changes were observed,and immunocytofluorescence staining was used to detect the expression of M2-specific phenotype marker CD163 on the cell surface.A co-culture system of M2-type TAM and human cholangiocarcinoma cell line QBC-939 was established,and the cells were divided into control group,M2 group and PI3Kγinhibitor group.After corresponding treatment,QBC-939 cells were collected.Transwell assay was used to detect cell migration and invasion ability,and immunocytofluorescence staining was used to observe the expressions of E-cadherin and Vimentin in cells.Western blotting was used to detect the protein expression of E-cadherin,N-cadherin,Vimentin,MMP2 and MMP9 in cells,and tumor cell spheroidization assay was used to detect cell stemness.Western blotting was used to detect the expression of cancer stem cell-related proteins CD133,OCT4 and SOX2 in the cells.[WTHZ]Results After induction by PMA and IL-4,the cells showed a typical M2 shape,and the CD163 fluorescence intensity increased significantly.However,after the intervention of PI3Kγinhibitor,the number of M2 cells was significantly reduced,and the fluorescence intensity of CD163 was weakened.Compared with the M2 group,the number of cells migrated and invaded in the PI3Kγinhibitor group decreased(P<0.05).The fluorescence density of E-cadherin increased,the fluorescence density of Vimentin decreased(P<0.05),and the expression level of E-cadherin protein was up-regulated.However,the protein expressions of N-cadherin,Vimentin,MMP2 and MMP9 were all down-regulated(P<0.05),at the same time,the number of tumor cells spheroids decreased(P<0.05),and the protein expres

关 键 词：胆管癌 磷酸肌醇3-激酶γ 肿瘤相关巨噬细胞 上皮-间质转化 干性 

分 类 号：R735.8[医药卫生—肿瘤]