IL-35在乳腺癌浸润淋巴细胞中表达的临床意义及其对乳腺癌细胞生物学功能的影响

发布时间：2018-04-04 20:16

本文选题：白细胞介素-35　切入点：调节性T细胞　出处：《山东大学》2016年博士论文

【摘要】：研究背景机体的免疫系统功能状态同恶性肿瘤的发生发展密切相关。2002年Schreiber和Dunn在肿瘤免疫监视理论的基础上提出了肿瘤免疫编辑学说(cancer immunoediting),认为免疫系统除了可以识别并清除肿瘤细胞,还具有免疫重塑功能,即对肿瘤细胞进行免疫选择,使免疫原性弱的肿瘤细胞进入免疫逃逸阶段,最终实现肿瘤进展。调节性T细胞(Treg)是免疫抑制中的重要一环,可以诱导肿瘤细胞通过免疫逃逸逃避效应细胞的识别和杀伤,促进肿瘤的发生发展。IL-35是2007年由Collison等和Niedbala等两个研究小组近乎同时发现的新型细胞因子,由IL-12的α亚基IL-12p35和IL-27的p亚基EBB以异二聚体形式组成。IL-35是主要来源于调节性T细胞(Treg)的抑制性细胞因子,它不仅可以直接促进Treg分化、增殖和发挥最大免疫调节作用,还可以诱导产生新型iTR35细胞,从而级联放大机体的免疫抑制调控网络,是促进肿瘤免疫逃逸的重要因素。新近的研究证明,除了调节性T细胞外,调节性B细胞、部分肿瘤细胞均可表达IL-35,提示IL-35同肿瘤的发生、发展关系密切。在胰腺癌、直肠癌等肿瘤的研究中,可检测到IL-35的表达,并且IL-35的高表达同不良的临床病理因素相关。第一部分IL-35在乳腺癌浸润淋巴细胞和血浆中的表达及其临床意义研究目的明确IL-35在乳腺癌患者中的表达情况,分析IL-35高表达与各临床病理因素之间的相关性,评估IL-35表达与患者预后转归的关联及能否作为乳腺癌新的生物标记物。研究方法入组2008年01月至2010年05月行手术切除的、具有完整随访资料的女性乳腺癌患者110例,应用免疫组织化学方法检测乳腺癌组织中IL-35表达情况,探讨IL-35表达同乳腺癌TNM分期、HER-2表达及无进展生存期(PFS)、总生存期(OS)的关系。入组2014年03月至2014年12月确诊的乳腺癌患者60例及健康对照30例,应用ELISA方法检测乳腺癌患者血浆中IL-35表达情况,探讨IL-35表达同乳腺癌TNM分期、ER、PR、HER-2、p53、EGFR、Ki67及分级的关系。研究结果在乳腺癌组织浸润淋巴细胞中发现IL-35表达,110例乳腺浸润性导管癌患者中,IL-35高表达者19例,占17.3%,低表达者48例,占43.6%,不表达者43例,占39.1%,IL-35在肿瘤浸润淋巴细胞中的表达水平在年龄大于50岁、术后病理分期Ⅲ期、肿块大于2cm及ER阴性患者中显著升高(p0.05)。Kaplan-Meier生存分析显示,肿瘤浸润淋巴细胞高表达IL-35的肿瘤患者组PFS和OS较低表达组显著缩短(p0.05)。乳腺癌患者血浆中IL-35水平高于健康对照组,但差别无统计学意义(0.24±0.11 ng/ml vs 0.23±0.10ng/ml, P=0.544)。但在乳腺癌患者中,Ⅲ期患者血浆IL-35表达水平显著高于Ⅰ和Ⅱ期的患者(0.29 ±0.13ng/ml VS 0.22±0.09 ng/ml, P=0.024),腋窝淋巴结阳性患者的血浆IL-35表达水平显著高于阴性患者(0.28±0.13 ng/ml VS 0.20±0.05ng/ml, P=0.004),但患者血浆IL-35表达水平与PR、HER-2、p53、EGFR、Ki67表达及分级无明显相关性(p0.05)。结论乳腺癌组织肿瘤浸润淋巴细胞中表达IL-35,高表达IL-35同多个预后不良因素密切相关,血浆中IL-35表达同临床分期和淋巴结转移密切相关。IL-35表达可以作为乳腺癌的不良预后因子。第二部分IL-35对乳腺肿瘤细胞生物学功能的影响及机制研究目的探讨IL-35对乳腺肿瘤细胞增殖、细胞周期、凋亡的影响及其分子机制。研究方法将外源性IL-35加入人乳腺癌细胞系MCF-7进行培养,应用MTT法检测增殖,流式细胞术检测细胞周期,比较实验组和对照组在增殖、凋亡及细胞周期中的差异。应用逆转录-PCR (RT-PCR)方法检测实验组和对照组促增殖相关分子(cyclin B、cyclin D、cyclin E、cdk2、cdk4)、抑制增殖相关分子(p15、 P18、p21、p27、p53)、促进凋亡相关分子(Fas、FasL、TRAIL、Bax)、抑制凋亡相关分子(FLIP、Bcl-2、survivin)的表达水平,比较两组表达差异。从转录水平探讨IL-35对乳腺癌细胞增殖和凋亡相关分子的影响。研究结果实验组增殖率较对照组显著提高,呈时间和浓度依赖性,提示IL-35在体外有促进乳腺癌细胞增殖的作用。与对照组相比,各实验组S期细胞的比例升高,浓度为0.2、10、50ng/ml组的S期细胞比例分别为：27.11%,29.17%,32.76%,34.80%,提示随着IL-35浓度升高,乳腺癌细胞增殖能力增强。与对照组相比,IL-35处理组的细胞增殖相关分子cyclin E、cdk2水平上调,抑制增殖相关分子p15水平下调,两组差别有统计学意义(p0.05)。IL-35处理组促进凋亡相关分子Fas、TRAIL水平下调(p0.05)。其他增殖及凋亡相关分子(cyclin B、 cyclin D、cdk4、p18、p21、p27、p53、FasL、Bax、 FLIP、Bc1-2、 survivin)水平变化实验组和对照组无显著差异(p0.05)。结论IL-35可以通过调控促增殖相关分子cyclin E、cdk2上调、抑制增殖相关分子p15下调、促进凋亡相关分子Fas、TRAIL下调,促进乳腺肿瘤细胞增殖,抑制肿瘤凋亡而促进肿瘤的免疫逃逸。IL-35可作为乳腺肿瘤治疗潜在的靶点。
[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.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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