慢性不可预知温和应激通过抑制JAK2-PI3K通路影响乳腺癌小鼠胸腺功能的实验研究  

Chronic Unpredictable Mild Stress Suppresses Thymus Function in Breast Cancer Mice by Inhibiting JAK2-PI3K Pathway

作　　者：郭文琴 许勰 刘曦 高建莉 GUO Wenqin;XU Xie;LIU Xi(School of Pharmaceutical Sciences,Zhejiang Chinese Medical University,Hangzhou(310053),China;Basic Medical School,Zhejiang Chinese Medical University)

机构地区：[1]浙江中医药大学药学院,杭州310053 [2]浙江中医药大学基础医学院

出　　处：《浙江中医药大学学报》2022年第4期376-387,共12页Journal of Zhejiang Chinese Medical University

基　　金：浙江省自然科学基金项目(LY22H160007);浙江省重点实验室项目(2012E10002);浙江中医药大学校级基金项目(2019ZG50JL)。

摘　　要：[目的]研究慢性应激导致的抑郁/焦虑等情绪失调状态下,乳腺癌机体胸腺免疫功能异常的神经-免疫学机制。[方法]将30只BALB/c小鼠随机分为正常组、模型组、慢性不可预知温和应激(chronic unpredictable mild stress,CUMS)-1组、CUMS-2组和CUMS-3组,每组6只。在小鼠乳腺脂肪垫接种4T1乳腺癌细胞,构建4T1乳腺癌模型,并采用不同程度CUMS刺激,分别构建不同程度CUMS乳腺癌小鼠模型。以旷场及强迫游泳实验观察行为活动;酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测大脑组织5-羟色胺(5-hydroxytryptamine,5-HT)水平;免疫荧光法观察胸腺上皮细胞(thymus epithelial cells,TECs)细胞角蛋白5(cytokeratin 5,CK5)和CK8的表达;流式细胞术分析胸腺细胞亚群分化簇3(cluster of differentiation 3,CD3)、CD4和CD8的比例;T细胞受体重排删除环(T cell receptor rearrangement excision circles,TRECs)法检测胸腺输出功能;实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,Real-time qPCR)分析胸腺基质淋巴细胞生成素(thymic stromal lymphopoietin,TSLP)、信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3)、白细胞介素-1α(interleukin-1α,IL-1α)、白细胞介素-7(interleukin-7,IL-7)、叉头样转录因子p3(forkhead transcription factor p3,Foxp3)、转化生长因子-β1(transforming growth factor-β1,TGF-β1)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的mRNA表达水平;免疫印迹法检测胸腺TSLP、STAT3、Janus激酶2(Janus kinase 2,JAK2)、磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase,PI3K)等相关蛋白表达水平。[结果]与正常组比较,模型组和CUMS各组胸腺结构紊乱,模型组胸腺组织TGF-β1、IL-5、IL-6、IL-10等mRNA水平显著增加,CD3+CD4-CD8-及CD3+CD4+CD8+T细胞比例显著降低,磷酸化信号转导和转录激活因子3(phospho-STAT3,p-STAT3)蛋白表达异常升高。与模型组比较,CUMS-3组小鼠大[Objective]To study the neuro-immunological mechanism of abnormal thymic immune function in breast cancer with emotional disorders such as depression/anxiety caused by chronic stress.[Methods]Thirty BALB/c mice were randomly divided into normal group,model group,chronic unpredictable mild stress(CUMS)-1 group,CUMS-2 group and CUMS-3 group,with 6 mice in each group.4T1 breast cancer cells were inoculated into mouse mammary fat pads to construct 4T1 breast cancer models,and 4T1 breast cancer model mice were stimulated with varying degrees of CUMS to build different degrees of CUMS breast cancer mouse models.The behavioral activities of mice were observed through open field test and forced swimming test;the level of 5-hydroxytryptamine(5-HT)in mouse brain was detected by enzyme-linked immunosorbent assay(ELISA);the expressions of cytokeratin 5(CK5)and cytokeratin 8(CK8)of thymus epithelial cells(TECs)were detected by immunofluorescence;the proportion of mouse thymic T cell subsets including cluster of differentia-tion 3(CD3),CD4,CD8 were analysis by flow cytometry;the function of thymic output was detected by T cell receptor rearrangement excision circles(TRECs)method;Real-time quantitative polymerase chain reaction(Real-time qPCR)was used to analysis mRNA levels of thymic stromal lymphopoietin(TSLP),signal transducer and activator of transcription 3(STAT3),interleukin-1α(IL-1α),interleukin-7(IL-7),forkhead transcription factor p3(Foxp3),transforming growth factor-β1(TGF-β1)and tumor necrosis factor-α(TNF-α),etc;the expression levels of TSLP,STAT3,Janus kinase 2(JAK2),phosphatidylinositol-3-kinase(PI3K)and other related proteins in the thymus were detected by Western blot.[Results]Compared with normal group,the structure of the thymus in model group and CUMS groups was disordered,the mRNA levels of TGF-β1,IL-5,IL-6,IL-10,etc.in the thymus tissue of model group were significantly increased,and the proportions of CD3+CD4-CD8-and CD3+CD4+CD8+T cells were significantly decreased,and phospho-STAT3(p-STAT3)was abno

关 键 词：慢性不可预知温和应激 情绪失调 乳腺癌 Janus激酶2-磷脂酰肌醇-3-激酶通路 胸腺功能 作用机制 

分 类 号：R331[医药卫生—人体生理学]