| 郭文琴,许勰,刘曦,等.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[J].浙江中医药大学学报,2022,46(4):376-387. |
| 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 |
| DOI：10.16466/j.issn1005-5509.2022.04.005 |
| 中文关键词: 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 |
| 英文关键词: chronic unpredictable mild stress emotional disorders breast cancer JAK2-PI3K pathway thymus function mechanism |
| 基金项目: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) |
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| 中文摘要: |
| [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组小鼠大脑5-HT水平明显降低，肺转移率增高，胸腺输出功能明显抑制，髓质中TECs数量显著减少，IL-5等多个细胞因子mRNA水平显著降低，TSLP和IL-7 mRNA水平显著升高，胸腺组织磷酸化Janus激酶2(phospho-JAK2，p-JAK2)、磷酸化磷脂酰肌醇-3-激酶p55(phospho-PI3K p55，p-PI3K p55)降低，差异均有统计学意义(P＜0.05)。[结论] 乳腺癌小鼠的胸腺功能紊乱，而CUMS可能通过抑制JAK2-PI3K通路，进一步抑制乳腺癌小鼠的胸腺功能，促进乳腺癌肺转移。 |
| 英文摘要: |
| [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 differentiation 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 abnormally elevated. Compared with model group， the level of 5-HT in the brain of CUMS-3 group was significantly decreased， the rate of lung metastasis was increased， the output function of the thymus was significantly inhibited， the number of TECs in the medulla was significantly decreased， IL-5 and other cytokine mRNA levels were significantly decreased， IL-7 and TSLP mRNA levels were significantly increased， the expressions of phospho-Janus kinase 2(p-JAK2) and phospho-phosphatidylinositol-3-kinase p55 (p-PI3K p55) in thymus tissue were statistically decreased， the differences were statistically significant (P＜0.05). [Conclusion] The thymus function of breast cancer mice is disordered， and CUMS may further inhibit the thymus function of breast cancer mice and promote lung metastasis by inhibiting the JAK2-PI3K pathway. |
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