趋化因子CCL20及细胞因子IL-32对肿瘤组织中Treg细胞迁移能力影响的研究

发布时间：2018-05-19 15:39

本文选题：非小细胞肺癌 + 趋化因子CCL20　；参考：《郑州大学》2016年博士论文

【摘要】：研究背景及目的肺癌是世界范围内较为常见的一种癌症,其发病率随着环境污染和人类生活方式的改变而成上升趋势。其中,非小细胞肺癌是临床常见肺癌类型,其发病率占总肺癌人群的85%左右。食管癌是全球范围内致死率较高的癌症之一,是一种致死性极高的消化道肿瘤,其致死率位居死亡诱因第8位,且手术后5年生存率仅为20-30%。尽管随着成像技术、手术以及放疗和化疗的进步,肺癌及食管癌的治疗取得一定的进展,但是存活率并未有明显提高,这使得该类癌症的治疗面临较大挑战。在人体内部,免疫系统的功能是识别和杀伤肿瘤细胞,然而,免疫系统的功能却受到了明显的抑制,大量的先天性和适应性免疫细胞参与了免疫抑制。在肿瘤免疫抑制微环境中,肿瘤细胞及肿瘤基质通过分泌趋化因子和细胞因子,招募Tregs、骨髓来源的抑制细胞(myeloid-derived suppressor cells,MDSCs)、Th17细胞和B-Reg细胞(regulatory B cells)等到达肿瘤组织,形成免疫抑制微环境,促进肿瘤的进展。研究表明,在许多类型原发或者转移肿瘤中如食管癌、卵巢癌、乳腺癌、肝癌、肺癌、口腔鳞癌、前列腺癌、皮肤鳞癌等,均可发现大量Tregs细胞的浸润,而且Tregs细胞水平的变化与肿瘤的预后密切相关,大量的Tregs细胞浸润,预示肿瘤的预后较差。多项研究结果显示,趋化因子在免疫细胞迁移运动过程中发挥重要作用,其中,趋化因子CCL20通过与其特异性受体CCR6结合,参与Treg细胞向肿瘤部位的迁移运动,从而在在多种癌症如结直肠癌和喉鳞状细胞癌等的发生、进展及远处转移过程中发挥重要作用,逐渐受到重视,然而,关于趋化因子CCL20在非小细胞肺癌进展中的作用却尚未揭示。CXCL8是一种重要的多功能细胞因子,可通过自分泌和旁分泌途径,与其受体CXCR1和CXCR2结合,在乳腺癌、肺癌、结直肠癌和黑色素瘤等的发生、发展过程中起到非常重要的作用。而CXCL8在食管癌发展过程中作用的有关研究较少。IL-32是一种重要的前炎症因子,在癌组织高表达,通过激活NF-k B通路,刺激细胞因子如CXCL8等的释放,促进癌症的发展。趋化因子CXCL8在多种癌症中参与调节性T细胞向肿瘤组织的定向迁移运动,而IL-32是否可通过作用于CXCL8而促进食管癌的发展的相关研究较少。本课题探讨趋化因子CCL20及细胞因子IL-32在肺小细胞癌及食管鳞癌组织招募Tregs细胞的相关机制,以期对其治疗提供新的方法与思路。第一部分趋化因子CCL20在非小细胞肺癌组织部位对Treg细胞迁移运动能力影响的研究1目的趋化因子CCL20在Treg细胞向非小细胞肺癌组织部位归巢过程中的分子机制的研究。2方法2.1 RT-PCR方法检测CCL20、CD4、Foxp3、IL-10在非小细胞肺癌组织或癌旁组织中的表达;2.2采用免疫组化方法在蛋白水平检测CCL20表达水平及其主要来源细胞;2.3流式细胞术检测非小细胞肺癌组织浸润淋巴细胞(TIL)和外周血PBMC中T细胞亚群比例及CCR6在T细胞亚群表达比例;2.4 ELISA检测化疗药物多西他赛处理或对照组细胞系A549对其分泌CCL20能力的影响;2.5统计分析非小细胞癌组织中CCL20表达水平与临床分期及生存期的影响。3结果3.1 RT-PCR和免疫组化结果均显示,趋化因子CCL20在非小细胞肺癌组织中表达水平高于相对应的癌旁组织;3.2非小细胞肺癌组织中,趋化因子CCL20与Treg细胞标志物CD4和Foxp3表达水平均呈正相关,且与炎性因子IL-10表达正相关;3.3流式细胞术结果显示,非小细胞肺癌组织部位TIL中Treg亚群比例远高于外周血PBMC,且CCR6+Treg细胞比例在TIL中比例高于PBMC;3.4趋化因子CCL20在临床终末期(III-IV期)非小细胞肺癌患者中表达水平高于早期患者(I-II期);3.5化疗药物多西他赛处理非小细胞肺癌细胞系降低了细胞系表达CCL20的水平。4结论趋化因子CCL20及其受体CCR6在非小细胞肺癌中可以增强Treg细胞向癌组织迁移运动的能力,化疗药物多西他赛可以降低CCL20的表达水平,可能具有降低Treg向癌组织迁移运动的能力。第二部分细胞因子IL-32在食管癌中的表达特征及其免疫学功能1目的研究IL-32在食管鳞癌组织招募Tregs细胞的相关机制,以期对该类疾病的治疗提供新的方法与思路。2方法2.1采用RT-PCR方法检测IL-32、Foxp3、CXCL8等基因标记分子在食管癌组织、癌旁组织或细胞系中的表达;2.2采用免疫组化法检测IL-32蛋白在食管癌组织中的表达水平;2.3采用siRNA技术敲除IL-32在食管癌细胞系TE1表达水平,并进一步检测其对CXCL8表达水平及Treg细胞迁移的影响;2.4流式细胞术检测Treg细胞在食管癌组织(TIL)和外周血中(PBMC)的分布情况。2.5采用SPSS17.0分析IL-32表达水平与食管癌患者临床数据及术后生存期间的相关性;3.结果3.1 RT-PCR结果显示,IL-32在食管癌组织中的表达高于癌旁组织;细胞系分析结果显示,IL-32在食管正常细胞(Het-1α)中表达水平较低,在食管癌细胞系中表达水平存在较大差异,在TE1中表达最高,而KYSE450和KYSE70中几乎无表达;3.2 RT-PCR结果显示,IL-32表达水平与趋化因子CXCL8表达呈正相关,而IL-32表达水平与浸润的Treg细胞呈正相关;3.3流式细胞术结果显示,食管癌肿瘤组织中Treg细胞比例远高于外周血中比例;且TIL中CXCR2+Treg细胞所占比例高于PBMC。3.4 si RNA敲除细胞系TE1中IL-32基因,CXCL8在基因水平和蛋白水平表达水平均降低,Treg细胞的迁移能力明显降低;3.5临床分析结果显示,IL-32表达水平与食管癌患者临床分期、分化水平均呈负相关,且发生淋巴结转移的患者表达IL-32的比例比未发生淋巴结转移的患者比例高;IL-32表达水平与食管癌患者术后生存期呈负相关。4小结细胞因子IL-32参与食管癌局部微环境的形成,通过促进趋化因子CXCL8的表达水平上调Treg细胞向肿瘤部位的迁移能力和比例。
[Abstract]:Background and objective lung cancer is one of the most common cancers worldwide. The incidence of lung cancer is rising with the change of environmental pollution and human lifestyle. Non small cell lung cancer is a common clinical type of lung cancer, and its incidence is about 85% of the total number of lung cancer. One of the most fatal digestive tract tumors is the eighth leading cause of death, and the 5 year survival rate is only 20-30%. after 5 years of surgery, although with progress in imaging, surgery and radiotherapy and chemotherapy, the treatment of lung and esophageal cancer has made some progress, but the survival rate has not been significantly improved, which makes this type of cancer. In the human body, the function of the immune system is to identify and kill the tumor cells. However, the function of the immune system is obviously suppressed. A large number of innate and adaptive immune cells are involved in the immunosuppression. In the microenvironment of tumor immunosuppression, the tumor cells and the tumor matrix are secreted by chemokines. And cytokines, recruitment of Tregs, bone marrow derived inhibitory cells (myeloid-derived suppressor cells, MDSCs), Th17 cells and B-Reg cells (regulatory B cells), etc. reach the tumor tissue and form an immunosuppressive microenvironment to promote the progress of the tumor. Liver cancer, lung cancer, oral squamous cell carcinoma, prostate cancer, and skin squamous cell carcinoma can detect a large number of Tregs cells, and the changes in Tregs cell level are closely related to the prognosis of the tumor. A large number of Tregs cells infiltrate, indicating that the prognosis of the tumor is poor. The chemokine CCL20, which combines with its specific receptor CCR6, participates in the migration of Treg cells to the tumor site, which plays an important role in the occurrence, progression and distant metastasis of cancer such as colorectal cancer and laryngeal squamous cell carcinoma. However, the chemokine CCL20 is not small. The role of cell lung cancer in progress has not revealed that.CXCL8 is an important multifunctional cytokine, which can play an unusually important role in the development of breast cancer, lung cancer, colorectal cancer and melanoma through autocrine and paracrine pathways, combined with its receptor CXCR1 and CXCR2, and CXCL8 is used in the development of esophageal cancer. .IL-32 is an important pro-inflammatory factor, which is an important pro-inflammatory factor in cancer tissue, which stimulates the release of cytokines such as CXCL8 by activating the NF-k B pathway and promoting the development of cancer. Chemokine CXCL8 participates in the directional migration of regulatory T cells to tumor tissues in a variety of cancers, and whether IL-32 can act on C through the action of IL-32. There are few related studies on the development of esophageal cancer. We discuss the mechanism of chemokine CCL20 and cytokine IL-32 in the recruitment of Tregs cells in small cell carcinoma of the lung and squamous cell carcinoma of the esophagus, in order to provide new methods and ideas for its treatment. The first part of chemokine CCL20 in the tissue site of non-small cell lung cancer to Treg cells Study of the influence of migration ability 1 the molecular mechanism of chemokine CCL20 in the homing process of Treg cells to non small cell lung cancer by.2 method 2.1 RT-PCR method to detect the expression of CCL20, CD4, Foxp3, IL-10 in non small cell lung cancer tissues or para cancerous tissues; 2.2 use immunohistochemical method to detect CCL at protein level 20 expression level and its main source cells; 2.3 flow cytometry was used to detect the proportion of T cell subsets in non small cell lung cancer tissue infiltrating lymphocytes (TIL) and peripheral blood PBMC, and the expression ratio of CCR6 in T cell subsets; 2.4 ELISA was used to detect the effect of A549 on the ability to secrete CCL20 in the chemotherapy drug docetaxel treatment or the control group, and 2.5 statistical scores. Analysis of the expression level of CCL20, clinical stage and life period in non small cell carcinoma tissue,.3 results 3.1 RT-PCR and immunohistochemical results all showed that the expression level of chemokine CCL20 in non-small cell lung cancer tissues was higher than that of the corresponding para cancerous tissue; 3.2 the chemokine CCL20 and Treg cell markers CD4 and Foxp in non-small cell lung cancer tissues. The expression level of 3 was positively correlated with the expression of inflammatory factor IL-10, and the results of 3.3 flow cytometry showed that the proportion of Treg subgroups in TIL of non small cell lung cancer was much higher than that of PBMC in peripheral blood, and the proportion of CCR6+Treg cells in TIL was higher than that of PBMC; 3.4 chemokine CCL20 at the end of clinical stage (III-IV stage) of non small cell lung cancer patients. The level of expression was higher than that of early patients (I-II phase); 3.5 chemotherapy drug docetaxel treatment of non small cell lung cancer cell lines reduced the level of CCL20 in cell lines.4 conclusion chemokine CCL20 and its receptor CCR6 can enhance the migration and movement of Treg cells to cancer tissue in non small cell lung cancer, and chemotherapy drug docetaxel can reduce C The expression level of CL20 may have the ability to reduce the migration of Treg to cancer tissue. Second the expression of cytokine IL-32 in esophageal cancer and its immunological function 1 aim to study the mechanism of IL-32 in the recruitment of Tregs cells in the squamous cell carcinoma of the esophagus, in order to provide new methods and ideas for the treatment of this kind of disease by means of the.2 method 2.1. RT-PCR method was used to detect the expression of IL-32, Foxp3, CXCL8 and other gene markers in esophageal cancer tissue, para cancer tissue or cell line. 2.2 immunohistochemistry was used to detect the expression level of IL-32 protein in esophageal cancer tissue; 2.3 siRNA technology was used to knock off IL-32 in the esophageal cancer cell line TE1 expression level, and to further detect its CXCL8 expression water. The influence of horizontal and Treg cell migration; 2.4 flow cytometry was used to detect the distribution of Treg cells in esophageal cancer tissue (TIL) and external Zhou Xuezhong (PBMC).2.5 using SPSS17.0 analysis of IL-32 expression level with clinical data of esophageal cancer patients and the correlation between postoperative survival; 3. results 3.1 RT-PCR results showed that the expression of IL-32 in esophageal cancer tissues was high. The results of cell line analysis showed that the expression level of IL-32 in the normal esophageal cells (Het-1 alpha) was lower, the expression level in the esophageal carcinoma cell lines was different, the expression in TE1 was the highest, while the expression in KYSE450 and KYSE70 was almost no expression. The 3.2 RT-PCR results showed that the expression level of IL-32 was positively correlated with the expression of chemokine CXCL8, and I. The expression level of L-32 was positively correlated with the infiltration of Treg cells, and the results of 3.3 flow cytometry showed that the proportion of Treg cells in the tumor tissues of the esophageal carcinoma was much higher than that in the peripheral blood, and the proportion of CXCR2+Treg cells in TIL was higher than the IL-32 gene in TE1 of the PBMC.3.4 Si RNA knockout cell line, and the CXCL8 in the gene level and protein level decreased. The migration ability of reg cells decreased significantly; 3.5 the results of clinical analysis showed that the level of IL-32 expression was negatively correlated with the clinical stage and the level of differentiation in the patients with esophageal cancer, and the proportion of IL-32 was higher in patients with lymph node metastasis than in the patients without lymph node metastasis; the level of IL-32 expression was negatively related to the survival period of the patients with esophageal cancer after operation. .4 nodule cytokine IL-32 participates in the formation of local microenvironment of esophageal cancer and up regulation of the migration ability and proportion of Treg cells to the tumor site by promoting the expression level of chemokine CXCL8.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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