本文選題：非小細(xì)胞肺癌 + 趨化因子CCL20�。� 參考：《鄭州大學(xué)》2016年博士論文

【摘要】：研究背景及目的肺癌是世界范圍內(nèi)較為常見(jiàn)的一種癌癥,其發(fā)病率隨著環(huán)境污染和人類生活方式的改變而成上升趨勢(shì)。其中,非小細(xì)胞肺癌是臨床常見(jiàn)肺癌類型,其發(fā)病率占總肺癌人群的85%左右。食管癌是全球范圍內(nèi)致死率較高的癌癥之一,是一種致死性極高的消化道腫瘤,其致死率位居死亡誘因第8位,且手術(shù)后5年生存率僅為20-30%。盡管隨著成像技術(shù)、手術(shù)以及放療和化療的進(jìn)步,肺癌及食管癌的治療取得一定的進(jìn)展,但是存活率并未有明顯提高,這使得該類癌癥的治療面臨較大挑戰(zhàn)。在人體內(nèi)部,免疫系統(tǒng)的功能是識(shí)別和殺傷腫瘤細(xì)胞,然而,免疫系統(tǒng)的功能卻受到了明顯的抑制,大量的先天性和適應(yīng)性免疫細(xì)胞參與了免疫抑制。在腫瘤免疫抑制微環(huán)境中,腫瘤細(xì)胞及腫瘤基質(zhì)通過(guò)分泌趨化因子和細(xì)胞因子,招募Tregs、骨髓來(lái)源的抑制細(xì)胞(myeloid-derived suppressor cells,MDSCs)、Th17細(xì)胞和B-Reg細(xì)胞(regulatory B cells)等到達(dá)腫瘤組織,形成免疫抑制微環(huán)境,促進(jìn)腫瘤的進(jìn)展。研究表明,在許多類型原發(fā)或者轉(zhuǎn)移腫瘤中如食管癌、卵巢癌、乳腺癌、肝癌、肺癌、口腔鱗癌、前列腺癌、皮膚鱗癌等,均可發(fā)現(xiàn)大量Tregs細(xì)胞的浸潤(rùn),而且Tregs細(xì)胞水平的變化與腫瘤的預(yù)后密切相關(guān),大量的Tregs細(xì)胞浸潤(rùn),預(yù)示腫瘤的預(yù)后較差。多項(xiàng)研究結(jié)果顯示,趨化因子在免疫細(xì)胞遷移運(yùn)動(dòng)過(guò)程中發(fā)揮重要作用,其中,趨化因子CCL20通過(guò)與其特異性受體CCR6結(jié)合,參與Treg細(xì)胞向腫瘤部位的遷移運(yùn)動(dòng),從而在在多種癌癥如結(jié)直腸癌和喉鱗狀細(xì)胞癌等的發(fā)生、進(jìn)展及遠(yuǎn)處轉(zhuǎn)移過(guò)程中發(fā)揮重要作用,逐漸受到重視,然而,關(guān)于趨化因子CCL20在非小細(xì)胞肺癌進(jìn)展中的作用卻尚未揭示。CXCL8是一種重要的多功能細(xì)胞因子,可通過(guò)自分泌和旁分泌途徑,與其受體CXCR1和CXCR2結(jié)合,在乳腺癌、肺癌、結(jié)直腸癌和黑色素瘤等的發(fā)生、發(fā)展過(guò)程中起到非常重要的作用。而CXCL8在食管癌發(fā)展過(guò)程中作用的有關(guān)研究較少。IL-32是一種重要的前炎癥因子,在癌組織高表達(dá),通過(guò)激活NF-k B通路,刺激細(xì)胞因子如CXCL8等的釋放,促進(jìn)癌癥的發(fā)展。趨化因子CXCL8在多種癌癥中參與調(diào)節(jié)性T細(xì)胞向腫瘤組織的定向遷移運(yùn)動(dòng),而IL-32是否可通過(guò)作用于CXCL8而促進(jìn)食管癌的發(fā)展的相關(guān)研究較少。本課題探討趨化因子CCL20及細(xì)胞因子IL-32在肺小細(xì)胞癌及食管鱗癌組織招募Tregs細(xì)胞的相關(guān)機(jī)制,以期對(duì)其治療提供新的方法與思路。第一部分趨化因子CCL20在非小細(xì)胞肺癌組織部位對(duì)Treg細(xì)胞遷移運(yùn)動(dòng)能力影響的研究1目的趨化因子CCL20在Treg細(xì)胞向非小細(xì)胞肺癌組織部位歸巢過(guò)程中的分子機(jī)制的研究。2方法2.1 RT-PCR方法檢測(cè)CCL20、CD4、Foxp3、IL-10在非小細(xì)胞肺癌組織或癌旁組織中的表達(dá);2.2采用免疫組化方法在蛋白水平檢測(cè)CCL20表達(dá)水平及其主要來(lái)源細(xì)胞;2.3流式細(xì)胞術(shù)檢測(cè)非小細(xì)胞肺癌組織浸潤(rùn)淋巴細(xì)胞(TIL)和外周血PBMC中T細(xì)胞亞群比例及CCR6在T細(xì)胞亞群表達(dá)比例;2.4 ELISA檢測(cè)化療藥物多西他賽處理或?qū)φ战M細(xì)胞系A(chǔ)549對(duì)其分泌CCL20能力的影響;2.5統(tǒng)計(jì)分析非小細(xì)胞癌組織中CCL20表達(dá)水平與臨床分期及生存期的影響。3結(jié)果3.1 RT-PCR和免疫組化結(jié)果均顯示,趨化因子CCL20在非小細(xì)胞肺癌組織中表達(dá)水平高于相對(duì)應(yīng)的癌旁組織;3.2非小細(xì)胞肺癌組織中,趨化因子CCL20與Treg細(xì)胞標(biāo)志物CD4和Foxp3表達(dá)水平均呈正相關(guān),且與炎性因子IL-10表達(dá)正相關(guān);3.3流式細(xì)胞術(shù)結(jié)果顯示,非小細(xì)胞肺癌組織部位TIL中Treg亞群比例遠(yuǎn)高于外周血PBMC,且CCR6+Treg細(xì)胞比例在TIL中比例高于PBMC;3.4趨化因子CCL20在臨床終末期(III-IV期)非小細(xì)胞肺癌患者中表達(dá)水平高于早期患者(I-II期);3.5化療藥物多西他賽處理非小細(xì)胞肺癌細(xì)胞系降低了細(xì)胞系表達(dá)CCL20的水平。4結(jié)論趨化因子CCL20及其受體CCR6在非小細(xì)胞肺癌中可以增強(qiáng)Treg細(xì)胞向癌組織遷移運(yùn)動(dòng)的能力,化療藥物多西他賽可以降低CCL20的表達(dá)水平,可能具有降低Treg向癌組織遷移運(yùn)動(dòng)的能力。第二部分細(xì)胞因子IL-32在食管癌中的表達(dá)特征及其免疫學(xué)功能1目的研究IL-32在食管鱗癌組織招募Tregs細(xì)胞的相關(guān)機(jī)制,以期對(duì)該類疾病的治療提供新的方法與思路。2方法2.1采用RT-PCR方法檢測(cè)IL-32、Foxp3、CXCL8等基因標(biāo)記分子在食管癌組織、癌旁組織或細(xì)胞系中的表達(dá);2.2采用免疫組化法檢測(cè)IL-32蛋白在食管癌組織中的表達(dá)水平;2.3采用siRNA技術(shù)敲除IL-32在食管癌細(xì)胞系TE1表達(dá)水平,并進(jìn)一步檢測(cè)其對(duì)CXCL8表達(dá)水平及Treg細(xì)胞遷移的影響;2.4流式細(xì)胞術(shù)檢測(cè)Treg細(xì)胞在食管癌組織(TIL)和外周血中(PBMC)的分布情況。2.5采用SPSS17.0分析IL-32表達(dá)水平與食管癌患者臨床數(shù)據(jù)及術(shù)后生存期間的相關(guān)性;3.結(jié)果3.1 RT-PCR結(jié)果顯示,IL-32在食管癌組織中的表達(dá)高于癌旁組織;細(xì)胞系分析結(jié)果顯示,IL-32在食管正常細(xì)胞(Het-1α)中表達(dá)水平較低,在食管癌細(xì)胞系中表達(dá)水平存在較大差異,在TE1中表達(dá)最高,而KYSE450和KYSE70中幾乎無(wú)表達(dá);3.2 RT-PCR結(jié)果顯示,IL-32表達(dá)水平與趨化因子CXCL8表達(dá)呈正相關(guān),而IL-32表達(dá)水平與浸潤(rùn)的Treg細(xì)胞呈正相關(guān);3.3流式細(xì)胞術(shù)結(jié)果顯示,食管癌腫瘤組織中Treg細(xì)胞比例遠(yuǎn)高于外周血中比例;且TIL中CXCR2+Treg細(xì)胞所占比例高于PBMC。3.4 si RNA敲除細(xì)胞系TE1中IL-32基因,CXCL8在基因水平和蛋白水平表達(dá)水平均降低,Treg細(xì)胞的遷移能力明顯降低;3.5臨床分析結(jié)果顯示,IL-32表達(dá)水平與食管癌患者臨床分期、分化水平均呈負(fù)相關(guān),且發(fā)生淋巴結(jié)轉(zhuǎn)移的患者表達(dá)IL-32的比例比未發(fā)生淋巴結(jié)轉(zhuǎn)移的患者比例高;IL-32表達(dá)水平與食管癌患者術(shù)后生存期呈負(fù)相關(guān)。4小結(jié)細(xì)胞因子IL-32參與食管癌局部微環(huán)境的形成,通過(guò)促進(jìn)趨化因子CXCL8的表達(dá)水平上調(diào)Treg細(xì)胞向腫瘤部位的遷移能力和比例。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R734.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Wen-Jun Chang;Yan Du;Xin Zhao;Li-Ye Ma;Guang-Wen Cao;;Inflammation-related factors predicting prognosis of gastric cancer[J];World Journal of Gastroenterology;2014年16期

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本文編號(hào)：1910664