發(fā)布時間：2018-05-05 23:04

  本文選題：胃癌 + 中性粒細(xì)胞��； 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：目的:采用二甲亞砜(DM SO)誘導(dǎo)人HL-60細(xì)胞分化建立中性粒細(xì)胞模型,探討胃癌細(xì)胞對中性粒細(xì)胞表型及功能影響,以及在胃癌發(fā)生發(fā)展中的作用及機(jī)制,并采用小鼠骨髓來源的原代中性粒細(xì)胞進(jìn)行體外實(shí)驗(yàn)驗(yàn)證,為證實(shí)腫瘤相關(guān)中性粒細(xì)胞促進(jìn)胃癌惡性進(jìn)展提供理論基礎(chǔ)和實(shí)驗(yàn)依據(jù),同時也為靶向腫瘤相關(guān)中性粒細(xì)胞治療胃癌提供新的策略和靶點(diǎn)。方法:(1)采用1.25%DM SO誘導(dǎo)HL-60細(xì)胞分化為中性粒細(xì)胞樣細(xì)胞(HL-60N)。將胃癌細(xì)胞與HL-60N共培養(yǎng),探究胃癌細(xì)胞生物學(xué)功能變化。Transwell遷移和基質(zhì)膠侵襲實(shí)驗(yàn)檢測胃癌細(xì)胞遷移、侵襲功能變化情況;MTT實(shí)驗(yàn)檢測胃癌細(xì)胞存活情況;細(xì)胞克隆形成實(shí)驗(yàn)檢測胃癌細(xì)胞增殖情況。(2)收集胃癌細(xì)胞條件培養(yǎng)基處理HL-60N,收集活化后HL-60 N上清,作用于胃癌細(xì)胞,探究胃癌細(xì)胞生物學(xué)功能變化。Transwell遷移和基質(zhì)膠侵襲實(shí)驗(yàn)檢測胃癌細(xì)胞遷移、侵襲功能變化情況;MTT實(shí)驗(yàn)檢測胃癌細(xì)胞存活情況;平板克隆實(shí)驗(yàn)檢測胃癌細(xì)胞增殖情況。(3)采用實(shí)時熒光定量R T-PCR檢測胃癌細(xì)胞共培養(yǎng)組和胃癌細(xì)胞條件培養(yǎng)基活化組HL-60N中炎癥因子IL-1β、IL-6、IL-8和TNF-α等基因表達(dá)情況;實(shí)時熒光定量R T-PC R檢測HL-60N共培養(yǎng)和活化HL-60N培養(yǎng)上清處理胃癌細(xì)胞EMT相關(guān)指標(biāo)E-cadheri n、Vimentin、ZEB-1、S lug與干性相關(guān)指標(biāo)Sox2、Oct 4和Nanog的基因表達(dá)情況;Western blot檢測胃癌細(xì)胞ERK信號通路活化情況;采用ERK信號通路抑制劑預(yù)處理胃癌細(xì)胞后,再用活化HL-60N培養(yǎng)上清處理,Transwell實(shí)驗(yàn)檢測胃癌細(xì)胞遷移、侵襲功能變化情況,實(shí)時熒光定量RT-PC R檢測胃癌細(xì)胞EMT相關(guān)指標(biāo)基因表達(dá)情況。(4)流式分選小鼠骨髓中性粒細(xì)胞,并用小鼠來源胃癌細(xì)胞MFC條件培養(yǎng)基處理,實(shí)時熒光定量R T-PCR檢測中性粒細(xì)胞IL-1β、IL-6和TNF-α基因表達(dá)情況;ERK信號通路抑制劑預(yù)處理小鼠胃癌細(xì)胞后,再用活化小鼠骨髓中性粒細(xì)胞培養(yǎng)上清處理,Transwell遷移實(shí)驗(yàn)和基質(zhì)膠侵襲實(shí)驗(yàn)檢測小鼠胃癌細(xì)胞遷移、侵襲功能變化情況。結(jié)果:(1)與HL-60N細(xì)胞共培養(yǎng)后,胃癌細(xì)胞遷移、侵襲能力增強(qiáng),而增殖能力無明顯變化。(2)胃癌細(xì)胞條件培養(yǎng)基活化的HL-60N培養(yǎng)上清促進(jìn)胃癌細(xì)胞遷移和侵襲,而對胃癌細(xì)胞增殖無顯著影響。(3)HL-60N與胃癌細(xì)胞共培養(yǎng)或經(jīng)胃癌細(xì)胞條件培養(yǎng)基處理后,炎癥因子IL-1β、IL-6、IL-8和TNF-α基因表達(dá)上調(diào)。與HL-60N共培養(yǎng)或活化HL-60N培養(yǎng)上清處理的胃癌細(xì)胞EMT指標(biāo)E-cadhe rin表達(dá)下調(diào),Vimentin、ZEB-1和Slug表達(dá)上調(diào),干性相關(guān)指標(biāo)Sox2、Oct4和Nanog表達(dá)也上調(diào)�；罨疕L-60N培養(yǎng)上清可激活胃癌細(xì)胞ERK信號通路,阻斷ERK信號通路顯著抑制活化HL-60N培養(yǎng)上清促進(jìn)胃癌細(xì)胞遷移和侵襲作用。(4)經(jīng)小鼠胃癌細(xì)胞條件培養(yǎng)基活化的小鼠骨髓中性粒細(xì)胞IL-1β、IL-6和TNF-α基因表達(dá)上調(diào),其培養(yǎng)上清促進(jìn)胃癌細(xì)胞遷移和侵襲,而阻斷ER K信號通路顯著抑制其作用。結(jié)論:中性粒細(xì)胞與胃癌細(xì)胞共培養(yǎng)或經(jīng)胃癌細(xì)胞條件培養(yǎng)基活化后,高表達(dá)炎癥因子,并通過活化ERK信號通路,誘導(dǎo)胃癌細(xì)胞發(fā)生EMT,進(jìn)而促進(jìn)其遷移和侵襲。中性粒細(xì)胞與胃癌細(xì)胞的相互作用可能是其促進(jìn)胃癌發(fā)生發(fā)展的重要機(jī)制,可能成為胃癌治療的新靶點(diǎn)。
[Abstract]:Objective: to establish a neutrophils model based on the differentiation of human HL-60 cells induced by two DM SO, and to explore the effect of gastric cancer cells on the phenotype and function of neutrophils, as well as the role and mechanism in the development of gastric cancer. The primary neutrophils of mice derived from bone marrow from mice were used to verify the tumor related neutrophils in vitro. Cells promote the malignant progression of gastric cancer to provide theoretical and experimental basis, and also provide new strategies and targets for tumor related neutrophils targeting gastric cancer. Methods: (1) 1.25%DM SO was used to induce HL-60 cells to differentiate into neutrophilic granulocyte like cells (HL-60N). Gastric cancer cells were co cultured with HL-60N to explore the biological work of gastric cancer cells. Can change.Transwell migration and matrix glue invasion test to detect the migration of gastric cancer cells, the change of invasion function, MTT test to detect the survival of gastric cancer cells, and cell clone formation test to detect the proliferation of gastric cancer cells. (2) collect the conditioned medium of gastric cancer cells to treat HL-60N, collect the activated HL-60 N supernatant, and act on gastric cancer cells. Investigate the biological function changes of gastric cancer cell.Transwell migration and matrix gel invasion test to detect the migration of gastric cancer cells, the change of invasion function, MTT test to detect the survival of gastric cancer cells, and test the proliferation of gastric cancer cells by flat clones. (3) detection of gastric cancer cell co culture and gastric cancer cell lines by real-time quantitative R T-PCR The expression of IL-1 beta, IL-6, IL-8 and TNF- alpha in the active group HL-60N, and real-time fluorescent quantitative R T-PC R to detect HL-60N co culture and activated HL-60N culture supernatant for gastric cancer cells. OT was used to detect the activation of ERK signaling pathway in gastric cancer cells; after pretreatment of gastric cancer cells with ERK signal pathway inhibitor, activated HL-60N culture supernatant was used, Transwell test was used to detect the migration of gastric cancer cells and the change of invasion function. Real-time quantitative fluorescence quantitative RT-PC R was used to detect the expression of EMT related genes in gastric cancer. (4) flow sorting Mice bone marrow neutrophils were treated with MFC conditioned medium of gastric cancer cells from mice, and real-time fluorescence quantitative R T-PCR was used to detect the expression of IL-1 beta, IL-6 and TNF- alpha in neutrophils; ERK signal pathway inhibitor pretreated mouse gastric cancer cells and then activated mouse bone marrow neutrophils culture supernatant and Transwell migrated. Results: (1) after co culture with HL-60N cells, the migration of gastric cancer cells, the enhanced invasion ability, and the proliferation ability were not significantly changed. (2) the activation of the conditioned medium of gastric cancer cell culture medium promoted the migration and invasion of gastric cancer cells, and the gastric cancer cells were treated with gastric cancer cells. There was no significant effect on proliferation. (3) the expression of IL-1 beta, IL-6, IL-8 and TNF- a was up regulated by co culture of HL-60N with gastric cancer cells or by gastric cancer cell conditioned medium. The E-cadhe Rin table of the EMT index of gastric cancer cells treated with HL-60N co culture or activated by HL-60N culture supernatant was down regulated and Vimentin, ZEB-1 and expressions were up regulated and dry sex related The expression of Sox2, Oct4 and Nanog also up-regulated. Activated HL-60N culture supernatant activates the ERK signaling pathway of gastric cancer cells, blocking ERK signaling pathway significantly inhibits activation of HL-60N culture supernatant to promote the migration and invasion of gastric cancer cells. (4) mouse bone marrow neutrophil IL-1 beta, IL-6 and TNF- a gene activated by mouse gastric cancer cell conditioned medium Up-regulated expression, the culture supernatant promotes the migration and invasion of gastric cancer cells, and blocking ER K signaling pathway significantly inhibits its role. Conclusion: neutrophils and gastric cancer cells co culture or after gastric cancer cell conditioned medium activation, high expression of inflammatory factors, and by activating the ERK signaling pathway to induce gastric cancer cells to occur EMT, and then promote its migration. The interaction between neutrophils and gastric cancer cells may be an important mechanism to promote the development of gastric cancer. It may become a new target for the treatment of gastric cancer.

【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.2

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本文編號：1849664