發(fā)布時(shí)間：2018-04-04 20:16

  本文選題：白細(xì)胞介素-35　切入點(diǎn)：調(diào)節(jié)性T細(xì)胞　出處：《山東大學(xué)》2016年博士論文

【摘要】：研究背景機(jī)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(guān)。2002年Schreiber和Dunn在腫瘤免疫監(jiān)視理論的基礎(chǔ)上提出了腫瘤免疫編輯學(xué)說(cancer immunoediting),認(rèn)為免疫系統(tǒng)除了可以識(shí)別并清除腫瘤細(xì)胞,還具有免疫重塑功能,即對(duì)腫瘤細(xì)胞進(jìn)行免疫選擇,使免疫原性弱的腫瘤細(xì)胞進(jìn)入免疫逃逸階段,最終實(shí)現(xiàn)腫瘤進(jìn)展。調(diào)節(jié)性T細(xì)胞(Treg)是免疫抑制中的重要一環(huán),可以誘導(dǎo)腫瘤細(xì)胞通過免疫逃逸逃避效應(yīng)細(xì)胞的識(shí)別和殺傷,促進(jìn)腫瘤的發(fā)生發(fā)展。IL-35是2007年由Collison等和Niedbala等兩個(gè)研究小組近乎同時(shí)發(fā)現(xiàn)的新型細(xì)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(diào)節(jié)性T細(xì)胞(Treg)的抑制性細(xì)胞因子,它不僅可以直接促進(jìn)Treg分化、增殖和發(fā)揮最大免疫調(diào)節(jié)作用,還可以誘導(dǎo)產(chǎn)生新型iTR35細(xì)胞,從而級(jí)聯(lián)放大機(jī)體的免疫抑制調(diào)控網(wǎng)絡(luò),是促進(jìn)腫瘤免疫逃逸的重要因素。新近的研究證明,除了調(diào)節(jié)性T細(xì)胞外,調(diào)節(jié)性B細(xì)胞、部分腫瘤細(xì)胞均可表達(dá)IL-35,提示IL-35同腫瘤的發(fā)生、發(fā)展關(guān)系密切。在胰腺癌、直腸癌等腫瘤的研究中,可檢測(cè)到IL-35的表達(dá),并且IL-35的高表達(dá)同不良的臨床病理因素相關(guān)。第一部分IL-35在乳腺癌浸潤(rùn)淋巴細(xì)胞和血漿中的表達(dá)及其臨床意義研究目的 明確IL-35在乳腺癌患者中的表達(dá)情況,分析IL-35高表達(dá)與各臨床病理因素之間的相關(guān)性,評(píng)估IL-35表達(dá)與患者預(yù)后轉(zhuǎn)歸的關(guān)聯(lián)及能否作為乳腺癌新的生物標(biāo)記物。研究方法入組2008年01月至2010年05月行手術(shù)切除的、具有完整隨訪資料的女性乳腺癌患者110例,應(yīng)用免疫組織化學(xué)方法檢測(cè)乳腺癌組織中IL-35表達(dá)情況,探討IL-35表達(dá)同乳腺癌TNM分期、HER-2表達(dá)及無進(jìn)展生存期(PFS)、總生存期(OS)的關(guān)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(duì)照30例,應(yīng)用ELISA方法檢測(cè)乳腺癌患者血漿中IL-35表達(dá)情況,探討IL-35表達(dá)同乳腺癌TNM分期、ER、PR、HER-2、p53、EGFR、Ki67及分級(jí)的關(guān)系。研究結(jié)果在乳腺癌組織浸潤(rùn)淋巴細(xì)胞中發(fā)現(xiàn)IL-35表達(dá),110例乳腺浸潤(rùn)性導(dǎo)管癌患者中,IL-35高表達(dá)者19例,占17.3%,低表達(dá)者48例,占43.6%,不表達(dá)者43例,占39.1%,IL-35在腫瘤浸潤(rùn)淋巴細(xì)胞中的表達(dá)水平在年齡大于50歲、術(shù)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(rùn)淋巴細(xì)胞高表達(dá)IL-35的腫瘤患者組PFS和OS較低表達(dá)組顯著縮短(p0.05)。乳腺癌患者血漿中IL-35水平高于健康對(duì)照組,但差別無統(tǒng)計(jì)學(xué)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(dá)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(jié)陽性患者的血漿IL-35表達(dá)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(dá)水平與PR、HER-2、p53、EGFR、Ki67表達(dá)及分級(jí)無明顯相關(guān)性(p0.05)。結(jié)論乳腺癌組織腫瘤浸潤(rùn)淋巴細(xì)胞中表達(dá)IL-35,高表達(dá)IL-35同多個(gè)預(yù)后不良因素密切相關(guān),血漿中IL-35表達(dá)同臨床分期和淋巴結(jié)轉(zhuǎn)移密切相關(guān)。IL-35表達(dá)可以作為乳腺癌的不良預(yù)后因子。第二部分IL-35對(duì)乳腺腫瘤細(xì)胞生物學(xué)功能的影響及機(jī)制研究目的探討IL-35對(duì)乳腺腫瘤細(xì)胞增殖、細(xì)胞周期、凋亡的影響及其分子機(jī)制。研究方法將外源性IL-35加入人乳腺癌細(xì)胞系MCF-7進(jìn)行培養(yǎng),應(yīng)用MTT法檢測(cè)增殖,流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期,比較實(shí)驗(yàn)組和對(duì)照組在增殖、凋亡及細(xì)胞周期中的差異。應(yīng)用逆轉(zhuǎn)錄-PCR (RT-PCR)方法檢測(cè)實(shí)驗(yàn)組和對(duì)照組促增殖相關(guān)分子(cyclin B、cyclin D、cyclin E、cdk2、cdk4)、抑制增殖相關(guān)分子(p15、 P18、p21、p27、p53)、促進(jìn)凋亡相關(guān)分子(Fas、FasL、TRAIL、Bax)、抑制凋亡相關(guān)分子(FLIP、Bcl-2、survivin)的表達(dá)水平,比較兩組表達(dá)差異。從轉(zhuǎn)錄水平探討IL-35對(duì)乳腺癌細(xì)胞增殖和凋亡相關(guān)分子的影響。研究結(jié)果實(shí)驗(yàn)組增殖率較對(duì)照組顯著提高,呈時(shí)間和濃度依賴性,提示IL-35在體外有促進(jìn)乳腺癌細(xì)胞增殖的作用。與對(duì)照組相比,各實(shí)驗(yàn)組S期細(xì)胞的比例升高,濃度為0.2、10、50ng/ml組的S期細(xì)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(xì)胞增殖能力增強(qiáng)。與對(duì)照組相比,IL-35處理組的細(xì)胞增殖相關(guān)分子cyclin E、cdk2水平上調(diào),抑制增殖相關(guān)分子p15水平下調(diào),兩組差別有統(tǒng)計(jì)學(xué)意義(p0.05)。IL-35處理組促進(jìn)凋亡相關(guān)分子Fas、TRAIL水平下調(diào)(p0.05)。其他增殖及凋亡相關(guān)分子(cyclin B、 cyclin D、cdk4、p18、p21、p27、p53、FasL、Bax、 FLIP、Bc1-2、 survivin)水平變化實(shí)驗(yàn)組和對(duì)照組無顯著差異(p0.05)。結(jié)論IL-35可以通過調(diào)控促增殖相關(guān)分子cyclin E、cdk2上調(diào)、抑制增殖相關(guān)分子p15下調(diào)、促進(jìn)凋亡相關(guān)分子Fas、TRAIL下調(diào),促進(jìn)乳腺腫瘤細(xì)胞增殖,抑制腫瘤凋亡而促進(jìn)腫瘤的免疫逃逸。IL-35可作為乳腺腫瘤治療潛在的靶點(diǎn)。
[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(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.9

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