本文選題：腦膠質(zhì)瘤　切入點(diǎn)：腫瘤細(xì)胞干性　出處：《鄭州大學(xué)》2017年碩士論文

【摘要】：目的腦膠質(zhì)瘤(Glioma)是顱內(nèi)常見的惡性腫瘤之一,發(fā)病率呈逐年上升趨勢(shì),且因易復(fù)發(fā),病人死亡率高。腦膠質(zhì)瘤干細(xì)胞(Glioma stem cells,GSCs)是一群決定腦膠質(zhì)瘤發(fā)生、發(fā)展、復(fù)發(fā)及耐藥的關(guān)鍵細(xì)胞,因此尋找影響腫瘤細(xì)胞干性的機(jī)制具有重要的理論意義和實(shí)用價(jià)值。由各種基質(zhì)細(xì)胞和免疫細(xì)胞及其分泌的細(xì)胞因子所組成的腫瘤微環(huán)境是GSCs存活和干性維持的基本條件。最新研究表明,腫瘤相關(guān)巨噬細(xì)胞(Tumor-associated macrophages,TAMs)和骨髓來源的抑制性細(xì)胞(Myeloid-derived suppressor cells,MDSCs)能夠通過分泌細(xì)胞因子促進(jìn)腫瘤細(xì)胞的干性,然而調(diào)節(jié)性T細(xì)胞(Regulatory T cells,Tregs)作為在腫瘤微環(huán)境部位發(fā)揮免疫抑制功能的一群重要免疫細(xì)胞,其與腦膠質(zhì)瘤干細(xì)胞之間的相互作用機(jī)制研究仍不清楚。本研究目的是探討Tregs與GSCs之間相互作用及詳細(xì)機(jī)制。方法收集2013年6月至2016年6月就診于鄭州大學(xué)第一附屬醫(yī)院的腦膠質(zhì)瘤患者癌組織與配對(duì)癌旁組織86對(duì),采用熒光定量法檢測(cè)CD133等基因表達(dá)水平;免疫組化法驗(yàn)證CD133在腫瘤組織中的高表達(dá);收取腦膠質(zhì)瘤患者的外周血,密度梯度離心法獲得外周血單個(gè)核細(xì)胞(Peripheral Blood Mononuclear Cells,PBMCs),磁珠分選法獲得純化的Treg細(xì)胞;Treg細(xì)胞分別與腦膠質(zhì)瘤細(xì)胞系U251、U87在Transwell板中共孵育;流式細(xì)胞術(shù)、熒光定量法和低粘附板成球法和檢測(cè)孵育后腫瘤細(xì)胞干性的變化;多因子法篩選細(xì)胞因子TGF-β、IL-6;熒光定量和免疫熒光鑒定TGF-β的來源;Western-Blot技術(shù)檢測(cè)信號(hào)通路上p-NF-κB、p-STAT3、CD133、SOX2等蛋白的表達(dá)水平,慢病毒轉(zhuǎn)染法構(gòu)建IL-6穩(wěn)定敲低的細(xì)胞系。結(jié)果1.CD133在腦膠質(zhì)瘤組織中表達(dá)明顯高于癌旁組織,并且CD133的表達(dá)與病人的生存和預(yù)后呈負(fù)相關(guān),CD133高表達(dá)的病人預(yù)后更差;2.Tregs能夠通過分泌TGF-β促進(jìn)腦膠質(zhì)瘤細(xì)胞U251和U87干性基因和成球能力上調(diào);3.TGF-β作用于腫瘤細(xì)胞后,通過激活ΝF-κB信號(hào)通路促進(jìn)腫瘤細(xì)胞自分泌IL-6;4.當(dāng)阻斷腫瘤細(xì)胞分泌IL-6時(shí),TGF-β促進(jìn)腫瘤細(xì)胞的干性的能力也明顯下調(diào)。并且IL-6的表達(dá)與病人的生存和預(yù)后呈負(fù)相關(guān),IL-6低表達(dá)的病人具有一個(gè)較好的預(yù)后;4.腫瘤細(xì)胞自分泌的IL-6通過激活STAT3信號(hào)通路從而促進(jìn)腫瘤細(xì)胞的干性。結(jié)論在腦膠質(zhì)瘤腫瘤微環(huán)境中,Treg通過分泌TGF-β促進(jìn)腫瘤細(xì)胞自分泌ΙL-6,從而促進(jìn)腫瘤細(xì)胞干性,TGF-β和IL-6均可作為腦膠質(zhì)瘤治療的潛在靶點(diǎn),為腦膠質(zhì)瘤的臨床治療提供了堅(jiān)實(shí)的理論依據(jù)。
[Abstract]:Objective Glioma (Glioma) is one of the most common malignant tumors in the brain. The incidence of Glioma is increasing year by year, and the mortality of patients is high because of its easy recurrence. Glioma stem cells are a group of gliomas that determine the occurrence and development of gliomas. Key cells for relapse and drug resistance, Therefore, it is of great theoretical significance and practical value to explore the mechanism of affecting the dryness of tumor cells. The tumor microenvironment composed of various stromal cells, immune cells and cytokines secreted by them is the survival and dry maintenance of GSCs. Basic conditions. Recent research shows that. Tumor-associated macrophages (Tumor-associated macrophages) and bone marrow-derived inhibitory cells (Myeloid-derived suppressor cells / MDSCs) can promote the dryness of tumor cells by secreting cytokines. However, regulatory T cells T cells Tregs are a group of important immune cells that play an immunosuppressive role in the tumor microenvironment. The purpose of this study was to explore the interaction between Tregs and GSCs and its detailed mechanism. Methods the first visit to Zhengzhou University from June 2013 to June 2016 was conducted. 86 pairs of brain glioma tissues and matched paracarcinomatous tissues in affiliated hospitals, Fluorescence quantitative method was used to detect the expression of CD133 and other genes, immunohistochemical method was used to verify the high expression of CD133 in tumor tissue, and peripheral blood was collected from glioma patients. Peripheral Blood Mononuclear cells of peripheral blood were obtained by density gradient centrifugation, and purified Treg cells were incubated with glioma cell line U251 U87 on Transwell plate, respectively. Fluorescence quantitative assay and low adhesion plate granulation assay were used to detect the changes of tumor cell dryness after incubation. The cytokines TGF- 尾 and IL-6 were screened by multifactor method. Western-Blot technique was used to detect the expression level of p-NF- 魏 Bmp-STAT3, CD133SOX2 and other proteins in the signaling pathway, and the origin of TGF- 尾 was identified by Western-Blot technique. IL-6 stable knockout cell lines were constructed by lentivirus transfection. Results the expression of 1.CD133 in gliomas was significantly higher than that in paracancerous tissues. Moreover, the expression of CD133 was negatively correlated with the survival and prognosis of the patients. The prognosis of patients with high expression of CD133 was even worse. 2. TGF- 尾 could promote the upregulation of the dry genes and the ability of granulation of glioma cells by secreting TGF- 尾. 3. TGF- 尾 acted on the tumor cells. The ability of TGF- 尾 to promote the dryness of tumor cells was also down-regulated when tumor cells were blocked from secreting IL-6. And the expression of IL-6 was negatively correlated with the survival and prognosis of patients. The autocrine IL-6 of tumor cells promotes the dryness of tumor cells by activating STAT3 signaling pathway. Conclusion in the microenvironment of glioma tumors, Treg promotes tumor cells by secreting TGF- 尾. Autocrine I L-6, thus promoting tumor cell dryness, TGF- 尾 and IL-6, may be potential targets for the treatment of glioma. It provides a solid theoretical basis for the clinical treatment of glioma.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.41

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