本文選題：宮頸癌 + IL-10��； 參考：《華北理工大學(xué)》2017年碩士論文

【摘要】：目的宮頸癌的發(fā)病趨勢已呈現(xiàn)低齡化。白介素-10(IL-10)作為一種具有免疫抑制作用的細胞因子對宮頸癌變的發(fā)生、發(fā)展具有重要作用。當(dāng)其在人體內(nèi)大量合成可抑制免疫系統(tǒng)的調(diào)控,引起HPV病毒入侵,導(dǎo)致宮頸癌的發(fā)生。在宮頸腫瘤生長的微環(huán)境中,腫瘤相關(guān)巨噬細胞(tumor associated macrophages,TAMs)通過分泌多種細胞因子參與腫瘤的生長、侵襲和遷移。TAMs作為表達IL-10的主要來源之一,其在宮頸癌變中調(diào)控IL-10表達的分子機制未曾報道。課題組前期研究已證實,宮頸腫瘤局部組織IL-10表達明顯升高,且該基因啟動子的低甲基化修飾促進其表達,這提示DNA甲基化修飾在應(yīng)對病毒入侵和局部炎癥環(huán)境的變化過程中具有重要影響。生物信息學(xué)分析顯示:E26轉(zhuǎn)錄因子-1(Ets1)和特異性蛋白1(Sp1)在IL-10基因啟動子上結(jié)合的順式作用元件含有CG位點,且有報道稱Ets1和Sp1的表達與宮頸癌的發(fā)生相關(guān),但其是否參與TAMs表達IL-10的調(diào)控機制及其在宮頸癌中的作用研究未見報道。因此,本研究利用人單核巨噬細胞、人宮頸癌細胞和人不同病理宮頸組織,探討TAMs中DNA甲基化參與Ets1和或Sp1介導(dǎo)IL-10基因轉(zhuǎn)錄調(diào)控的分子機制及其在宮頸癌變中的作用,旨在為以TAMs作為靶點的宮頸癌靶向治療提供新思路。方法選取2014年9月至2016年3月就診于唐山市工人醫(yī)院并行宮頸活檢及手術(shù)治療患者160例,其中正常宮頸47例(對照組)、子宮頸上皮內(nèi)瘤變Ⅰ級27例(CINⅠ組)、子宮頸上皮內(nèi)瘤變Ⅱ-Ⅲ級46例(CINⅡ-Ⅲ組)和宮頸鱗癌40例(CSCC組)。在線查看所有患者的電子病例并了解患者的基本信息情況。采用免疫雙熒光染色檢測各組人宮頸組織中IL-10、Ets1和Sp1分別同CD68的共表達;臨床分析IL-10+TAMs、Ets1+TAMs和Sp1+TAMs水平與宮頸鱗癌臨床病理特征的關(guān)系;采用人單核細胞THP-1體外誘導(dǎo)形成宮頸TAMs;利用si RNA干擾以及q RT-PCR和Western blot技術(shù)分析TAMs中轉(zhuǎn)錄因子Ets1和(或)Sp1對IL-10基因表達水平的影響;生物信息學(xué)分析轉(zhuǎn)錄因子Sp1和Ets1在IL-10基因啟動子近端2000bp范圍內(nèi)可能的結(jié)合位點,并通過對TAMs甲基化干預(yù)、熒光素酶報告基因、染色質(zhì)免疫沉淀(CHIP)和甲基化特異性PCR(MSP)等分子生物學(xué)方法分析在宮頸TAMs中DNA甲基化對轉(zhuǎn)錄因子Sp1、Ets1與IL-10基因啟動子的結(jié)合及其轉(zhuǎn)錄活性的影響。結(jié)果1對照組、CINⅠ組、CINⅡ-Ⅲ組和CSCC組患者的基本信息(包括:年齡、初潮年齡、月經(jīng)周期、經(jīng)期天數(shù)、懷孕次數(shù)、流產(chǎn)次數(shù)、吸煙情況、飲酒情況)比較,差異均無統(tǒng)計學(xué)意義(P0.05)。2免疫雙熒光染色檢測不同病理宮頸組織中IL-10、Ets1和Sp1與TAMs的共表達結(jié)果顯示,伴隨宮頸病變的進展,宮頸組織中TAMs以及IL-10+TAMs、Ets1+TAMs和Sp1+TAMs的表達百分比均逐漸增加,且與正常組相比,CINⅠ組、CINⅡ-Ⅲ組和CSCC組中的表達逐漸升高,差異有統(tǒng)計學(xué)意義(P0.05)。3在CSCC組織中IL-10+TAMs、Ets1+TAMs和Sp1+TAMs的表達水平與年齡、腫瘤大小無關(guān)(P0.05);與分化程度、FIGO分期和淋巴結(jié)轉(zhuǎn)移有關(guān)(P0.05)。4在TAMs中采用干擾si RNA敲低Ets1、Sp1或同時敲低Ets1和Sp1基因表達,q RT-PCR和Western blot實驗結(jié)果顯示:敲低Sp1或同時敲低Ets1和Sp1基因均可下調(diào)IL-10的表達,且雙敲抑制其表達更為明顯,而僅敲低Ets1對IL-10的表達影響較小。5在甲基化干預(yù)實驗以及MSP測序和Ch IP實驗結(jié)果顯示,在TAMs中IL-10基因啟動子區(qū)的去甲基化促使轉(zhuǎn)錄因子Sp1與其結(jié)合并上調(diào)IL-10的表達。6進一步CHIP和免疫熒光染色實驗結(jié)果表明,Ets1在Sp1調(diào)控IL-10基因啟動子活性過程中起協(xié)同作用。結(jié)論1 TAMs源IL-10的表達與宮頸癌變呈正相關(guān)性。2在宮頸癌中轉(zhuǎn)錄因子Ets1和Sp1的表達上調(diào)且與TAMs源IL-10的表達相關(guān)。3在TAMs中Sp1結(jié)合位點DNA去甲基化在調(diào)控IL-10基因啟動子活性及其在宮頸癌變發(fā)生中具有重要促進作用,而Ets1表達促進Sp1調(diào)控IL-10基因啟動子的轉(zhuǎn)錄活性。
[Abstract]:Objective the incidence of cervical cancer has been reduced. Interleukins -10 (IL-10), as a cytokine with immunosuppressive effects, plays an important role in the development of cervical cancer. It is an important role in the development of cervical cancer. When it is widely synthesized in human body, the regulation of immunosuppressive immune system, the invasion of HPV virus, the occurrence of cervical cancer and the growth of cervical cancer. In the microenvironment, tumor associated macrophages (TAMs) is involved in the growth of tumor by secreting a variety of cytokines, invasion and migration of.TAMs as one of the main sources of IL-10 expression. The molecular mechanism of the regulation of IL-10 expression in cervical cancer has not been reported. The expression of IL-10 in the tissue is significantly elevated, and the gene promoter's low methylation modification promotes its expression. This suggests that DNA methylation modification plays an important role in response to changes in virus invasion and local inflammatory environment. Bioinformatics analysis shows that the E26 transcription factor -1 (Ets1) and specific protein 1 (Sp1) are on the IL-10 gene promoter. The combination of cis acting elements contains CG loci, and it is reported that the expression of Ets1 and Sp1 is related to the occurrence of cervical cancer, but it is not reported whether it participates in the regulatory mechanism of TAMs expression IL-10 and its role in cervical cancer. Therefore, this study used human mononuclear macrophages, human cervical cancer cells and different pathological cervical tissues. To discuss the molecular mechanism of DNA methylation involved in Ets1 and or Sp1 mediated transcription regulation of IL-10 gene in TAMs and its role in cervical cancer. The aim is to provide a new idea for the targeting therapy of cervical cancer with TAMs as a target. Methods 160 cases of cervical biopsy and surgical treatment in Tangshan City workers' hospital from September 2014 to March 2016 were selected. 47 cases of normal cervix (control group), 27 cases of cervical intraepithelial neoplasia (group CIN I), 46 cases of cervical intraepithelial neoplasia II - III (group CIN II - III) and 40 cases of cervical squamous cell carcinoma (group CSCC). The electronic cases of all patients were examined online and the basic information of the patients was understood. The cervical tissue of each group was detected by immunofluorescence staining. IL-10, Ets1 and Sp1 were co expressed with CD68, and the relationship between IL-10+TAMs, Ets1+TAMs and Sp1+TAMs levels with the clinicopathological features of cervical squamous cell carcinoma was analyzed. Human mononuclear cell THP-1 was induced to form cervical TAMs in vitro, and Si RNA interference and the transcriptional factor and / or transcriptional factors were analyzed. The effect of expression level; bioinformatics analysis of the possible binding sites of transcription factors Sp1 and Ets1 in the near end 2000bp of IL-10 gene promoter, and the analysis of DNA methyl in cervical TAMs by molecular biological methods such as TAMs methylation intervention, luciferase reporter gene, chromatin immunoprecipitation (CHIP) and methylation specific PCR (MSP). The effect of the combination of transcription factor Sp1, Ets1 and IL-10 gene promoter and its transcriptional activity. Results the basic information of 1 control groups, CIN I group, CIN II - III group and CSCC group (including age, menarche age, menstrual cycle, menstrual period, pregnancy times, abortion number, smoking, drinking) were not statistically significant (P 0.05) the co expression of IL-10, Ets1, Sp1 and TAMs in different pathological cervix tissues by.2 immunofluorescence staining showed that the percentage of TAMs, IL-10+TAMs, Ets1+TAMs and Sp1+TAMs in cervical tissues increased gradually with the progression of cervical lesions, and the expression of TAMs in the CIN I group, CIN II - III group and CSCC group compared with the normal group. The difference was statistically significant (P0.05), the expression level of IL-10+TAMs, Ets1+TAMs and Sp1+TAMs in the CSCC tissues was not related to the size of the tumor (P0.05) and the degree of differentiation, FIGO staging and lymph node metastasis (P0.05).4 in TAMs. Blot experimental results showed that the expression of IL-10 was downregulated by knocking low Sp1 or at the same time knocking at low Ets1 and Sp1 genes, and the expression of double knockdown inhibition was more obvious, but only the expression of low Ets1 on the expression of IL-10 had less influence on.5 in the methylation intervention experiment and the results of MSP sequencing and Ch IP experiment showed that the demethylation of the promoter region of the gene promoted the transfer in TAMs. Transcription factor Sp1 is combined with and up regulation of IL-10 expression.6 further CHIP and immunofluorescence staining experiments show that Ets1 plays a synergistic role in the Sp1 regulation of IL-10 gene promoter activity. Conclusion the expression of IL-10 in 1 TAMs source is positively correlated with cervical cancer,.2 is up regulation of Ets1 and Sp1 expressions in cervical cancer and is up to the source. 0 the expression of.3 related to the Sp1 binding site DNA demethylation in TAMs plays an important role in regulating IL-10 gene promoter activity and in cervical carcinogenesis, and Ets1 expression promotes the transcriptional activity of the IL-10 gene promoter by Sp1.
【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.33

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相關(guān)期刊論文 前10條

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本文編號：1970255