本文選題：白細胞介素-35　切入點：調節(jié)性T細胞　出處：《山東大學》2016年博士論文

【摘要】：研究背景機體的免疫系統(tǒng)功能狀態(tài)同惡性腫瘤的發(fā)生發(fā)展密切相關。2002年Schreiber和Dunn在腫瘤免疫監(jiān)視理論的基礎上提出了腫瘤免疫編輯學說(cancer immunoediting),認為免疫系統(tǒng)除了可以識別并清除腫瘤細胞,還具有免疫重塑功能,即對腫瘤細胞進行免疫選擇,使免疫原性弱的腫瘤細胞進入免疫逃逸階段,最終實現(xiàn)腫瘤進展。調節(jié)性T細胞(Treg)是免疫抑制中的重要一環(huán),可以誘導腫瘤細胞通過免疫逃逸逃避效應細胞的識別和殺傷,促進腫瘤的發(fā)生發(fā)展。IL-35是2007年由Collison等和Niedbala等兩個研究小組近乎同時發(fā)現(xiàn)的新型細胞因子,由IL-12的α亞基IL-12p35和IL-27的p亞基EBB以異二聚體形式組成。IL-35是主要來源于調節(jié)性T細胞(Treg)的抑制性細胞因子,它不僅可以直接促進Treg分化、增殖和發(fā)揮最大免疫調節(jié)作用,還可以誘導產生新型iTR35細胞,從而級聯(lián)放大機體的免疫抑制調控網(wǎng)絡,是促進腫瘤免疫逃逸的重要因素。新近的研究證明,除了調節(jié)性T細胞外,調節(jié)性B細胞、部分腫瘤細胞均可表達IL-35,提示IL-35同腫瘤的發(fā)生、發(fā)展關系密切。在胰腺癌、直腸癌等腫瘤的研究中,可檢測到IL-35的表達,并且IL-35的高表達同不良的臨床病理因素相關。第一部分IL-35在乳腺癌浸潤淋巴細胞和血漿中的表達及其臨床意義研究目的 明確IL-35在乳腺癌患者中的表達情況,分析IL-35高表達與各臨床病理因素之間的相關性,評估IL-35表達與患者預后轉歸的關聯(lián)及能否作為乳腺癌新的生物標記物。研究方法入組2008年01月至2010年05月行手術切除的、具有完整隨訪資料的女性乳腺癌患者110例,應用免疫組織化學方法檢測乳腺癌組織中IL-35表達情況,探討IL-35表達同乳腺癌TNM分期、HER-2表達及無進展生存期(PFS)、總生存期(OS)的關系。入組2014年03月至2014年12月確診的乳腺癌患者60例及健康對照30例,應用ELISA方法檢測乳腺癌患者血漿中IL-35表達情況,探討IL-35表達同乳腺癌TNM分期、ER、PR、HER-2、p53、EGFR、Ki67及分級的關系。研究結果在乳腺癌組織浸潤淋巴細胞中發(fā)現(xiàn)IL-35表達,110例乳腺浸潤性導管癌患者中,IL-35高表達者19例,占17.3%,低表達者48例,占43.6%,不表達者43例,占39.1%,IL-35在腫瘤浸潤淋巴細胞中的表達水平在年齡大于50歲、術后病理分期Ⅲ期、腫塊大于2cm及ER陰性患者中顯著升高(p0.05)。Kaplan-Meier生存分析顯示,腫瘤浸潤淋巴細胞高表達IL-35的腫瘤患者組PFS和OS較低表達組顯著縮短(p0.05)。乳腺癌患者血漿中IL-35水平高于健康對照組,但差別無統(tǒng)計學意義(0.24±0.11 ng/ml vs 0.23±0.10ng/ml, P=0.544)。但在乳腺癌患者中,Ⅲ期患者血漿IL-35表達水平顯著高于Ⅰ和Ⅱ期的患者(0.29 ±0.13ng/ml VS 0.22±0.09 ng/ml, P=0.024),腋窩淋巴結陽性患者的血漿IL-35表達水平顯著高于陰性患者(0.28±0.13 ng/ml VS 0.20±0.05ng/ml, P=0.004),但患者血漿IL-35表達水平與PR、HER-2、p53、EGFR、Ki67表達及分級無明顯相關性(p0.05)。結論乳腺癌組織腫瘤浸潤淋巴細胞中表達IL-35,高表達IL-35同多個預后不良因素密切相關,血漿中IL-35表達同臨床分期和淋巴結轉移密切相關。IL-35表達可以作為乳腺癌的不良預后因子。第二部分IL-35對乳腺腫瘤細胞生物學功能的影響及機制研究目的探討IL-35對乳腺腫瘤細胞增殖、細胞周期、凋亡的影響及其分子機制。研究方法將外源性IL-35加入人乳腺癌細胞系MCF-7進行培養(yǎng),應用MTT法檢測增殖,流式細胞術檢測細胞周期,比較實驗組和對照組在增殖、凋亡及細胞周期中的差異。應用逆轉錄-PCR (RT-PCR)方法檢測實驗組和對照組促增殖相關分子(cyclin B、cyclin D、cyclin E、cdk2、cdk4)、抑制增殖相關分子(p15、 P18、p21、p27、p53)、促進凋亡相關分子(Fas、FasL、TRAIL、Bax)、抑制凋亡相關分子(FLIP、Bcl-2、survivin)的表達水平,比較兩組表達差異。從轉錄水平探討IL-35對乳腺癌細胞增殖和凋亡相關分子的影響。研究結果實驗組增殖率較對照組顯著提高,呈時間和濃度依賴性,提示IL-35在體外有促進乳腺癌細胞增殖的作用。與對照組相比,各實驗組S期細胞的比例升高,濃度為0.2、10、50ng/ml組的S期細胞比例分別為：27.11%,29.17%,32.76%,34.80%,提示隨著IL-35濃度升高,乳腺癌細胞增殖能力增強。與對照組相比,IL-35處理組的細胞增殖相關分子cyclin E、cdk2水平上調,抑制增殖相關分子p15水平下調,兩組差別有統(tǒng)計學意義(p0.05)。IL-35處理組促進凋亡相關分子Fas、TRAIL水平下調(p0.05)。其他增殖及凋亡相關分子(cyclin B、 cyclin D、cdk4、p18、p21、p27、p53、FasL、Bax、 FLIP、Bc1-2、 survivin)水平變化實驗組和對照組無顯著差異(p0.05)。結論IL-35可以通過調控促增殖相關分子cyclin E、cdk2上調、抑制增殖相關分子p15下調、促進凋亡相關分子Fas、TRAIL下調,促進乳腺腫瘤細胞增殖,抑制腫瘤凋亡而促進腫瘤的免疫逃逸。IL-35可作為乳腺腫瘤治療潛在的靶點。
[Abstract]:The occurrence and development of immune system function on the background of the body with malignant tumors is closely related to.2002 Schreiber and Dunn in tumor immune surveillance theory proposed cancer immunoedting theory (cancer immunoediting), in addition to think that the immune system can recognize and eliminate tumor cells, also has the function of immune remodeling, immune selection of tumor the cells, weak immunogenicity of tumor cells into the immune escape stage, finally realize the tumor progression. Regulatory T cells (Treg) is an important part of the immune suppression, can induce tumor cells to escape immune escape through the recognition and killing effector cells, promote tumor occurrence and development of.IL-35 is a novel cytokine by 2007 two research teams such as Collison and Niedbala found that nearly at the same time, by the alpha subunit of IL-12 IL-12p35 and IL-27 P subunit EBB ISO two dimer form group .IL-35 is the main source in regulatory T cells (Treg) inhibitory cytokines, it can not only directly promote Treg differentiation, proliferation and play a role in regulating the maximum immunity, can induce the production of new iTR35 cells, immune suppression and cascade regulation network is an important factor to promote tumor immune escape. Recent research shows that in addition to regulatory T cells, regulatory B cells, some tumor cells could express IL-35, suggesting that IL-35 with tumor occurrence, development are closely related. In pancreatic cancer, colorectal cancer and other tumors, detected the expression of IL-35, and the high expression of IL-35 with clinical pathological factors related to poor. The first part of the expression of IL-35 in breast cancer invasion and the clinical significance of the expression of lymphocyte and plasma to clear IL-35 in patients with breast cancer and analysis of high expression of IL-35 with clinical disease The correlation between the physical factors, to assess the expression of IL-35 related outcome and the prognosis of patients and can be used as new biomarkers for breast cancer. Research methods into the group in 2008 01 to 2010 05 months underwent surgical resection, 110 female breast cancer patients with complete follow-up data of patients, the expression of IL-35 by immunohistochemical method in breast cancer, to investigate the expression of IL-35 with the TNM staging of breast cancer, the expression of HER-2 and progression free survival (PFS), overall survival (OS). The relationship between the group in 2014 03 months to December 2014 were 60 cases of breast cancer patients and 30 healthy controls, the expression of IL-35 in plasma was measured by ELISA method to investigate the expression of IL-35 in breast cancer with breast cancer TNM staging, ER, PR, HER-2, p53, EGFR, Ki67 and grading. The research results in breast cancer infiltrating lymphocytes found in the expression of IL-35, 110 cases of breast invasive ductal cancer In the high expression of IL-35 in 19 cases, accounting for 17.3%, 48 patients with low expression, no expression accounted for 43.6%, 43 cases, accounting for 39.1%, IL-35 expression level in tumor infiltrating lymphocytes in older than 50, postoperative pathological stage III, with more than 2cm and ER negative patients increased significantly (P0.05).Kaplan-Meier lymphocyte survival analysis showed that tumors with high expression of PFS and OS group IL-35 low expression group was significantly shortened the tumor (P0.05). The plasma level of IL-35 in breast cancer patients than in healthy controls, but the difference was not statistically significant (0.24 + 0.11 ng/ml vs + 0.23 0.10ng/ml, P=0.544). But in breast cancer patients, patients with plasma level of IL-35 was significantly higher than that in stage I and II patients (0.29 + 0.13ng/ml VS 0.22 + 0.09 ng/ml, P=0.024), the expression level was significantly higher than that of patients with negative plasma IL-35 patients with positive axillary lymph node (0.28 + 0.13 ng/ml VS 0.20 + 0.05ng/ml, P=0.004), but the plasma level IL-35 and PR, HER-2, p53, EGFR, Ki67 expression and classification (P0.05). There was no significant correlation between the expression of IL-35 in lymphocytes of tumor infiltrating breast cancer conclusion, the high expression of IL-35 with a number of adverse factors closely related to the prognosis, the expression of IL-35 with clinical stage and lymph node metastasis closely related to expression of.IL-35 can be considered as a poor prognostic factor of breast cancer in plasma. Objective to study the effect on breast cancer cell biological function and mechanism of the second part of the IL-35 of IL-35 on the proliferation of breast cancer cells, cell cycle, apoptosis and its molecular mechanism. Methods the exogenous IL-35 into human breast cancer cell line MCF-7 were cultured. Application of MTT method to detect the proliferation and cell cycle were detected by flow cytometry, comparing the experimental group and control group on proliferation, apoptosis and cell cycle differences. Reverse transcription -PCR (R T-PCR) method to detect the proliferation of the experimental group and the control group (cyclin B, cyclin molecular D, cyclin E, CDK2, CDK4), inhibit the proliferation of related molecules (p15, P18, p21, p27, p53), apoptosis related molecules (Fas, FasL, TRAIL, Bax), inhibition of apoptosis related molecules (FLIP, Bcl-2, survivin) expression levels, the difference between the two groups. To investigate the effect of IL-35 expression on proliferation and apoptosis of breast cancer cells and related molecules at the transcription level. The research results in the experimental group, the proliferation rate was significantly higher than the control group, in a time and concentration dependent, suggesting that IL-35 could promote the proliferation of breast cancer cells in vitro compared with the control group, the experimental group the proportion of cells in S phase increased, the concentration of 0.2,10,50ng/ml group the proportion of cells in S phase were 27.11%, 29.17%, 32.76%, 34.80%, suggesting that with increasing concentrations of IL-35, breast cancer cell proliferation ability. Compared with the control group, IL-35 treatment group cells The proliferation of related molecules cyclin and E, increases the level of CDK2, inhibit the proliferation of related molecules and decreased levels of p15, there was a significant difference between the two groups (P0.05) of.IL-35 group to promote apoptosis related molecules Fas, downregulation of TRAIL (P0.05). The proliferation and apoptosis related molecules (cyclin B, cyclin D, CDK4, P18, p21, p27. P53, FasL, Bax, FLIP, Bc1-2, survivin) there was no significant difference between the levels of the experimental group and the control group (P0.05). Conclusion IL-35 can regulate the proliferation related protein cyclin E, upregulation of CDK2, downregulation of p15 inhibits the proliferation of related molecules, apoptosis related molecules Fas, downregulation of TRAIL, promote the proliferation of breast cancer cells, target inhibiting the tumor apoptosis and promote tumor immune escape of.IL-35 can be used as a potential treatment of breast cancer.

【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.9

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