中性粒细胞激活ERK信号通路诱导EMT促进胃癌细胞迁移和侵袭

发布时间：2018-05-05 23:04

本文选题：胃癌 + 中性粒细胞　；参考：《江苏大学》2017年硕士论文

【摘要】：目的:采用二甲亚砜(DM SO)诱导人HL-60细胞分化建立中性粒细胞模型,探讨胃癌细胞对中性粒细胞表型及功能影响,以及在胃癌发生发展中的作用及机制,并采用小鼠骨髓来源的原代中性粒细胞进行体外实验验证,为证实肿瘤相关中性粒细胞促进胃癌恶性进展提供理论基础和实验依据,同时也为靶向肿瘤相关中性粒细胞治疗胃癌提供新的策略和靶点。方法:(1)采用1.25%DM SO诱导HL-60细胞分化为中性粒细胞样细胞(HL-60N)。将胃癌细胞与HL-60N共培养,探究胃癌细胞生物学功能变化。Transwell迁移和基质胶侵袭实验检测胃癌细胞迁移、侵袭功能变化情况;MTT实验检测胃癌细胞存活情况;细胞克隆形成实验检测胃癌细胞增殖情况。(2)收集胃癌细胞条件培养基处理HL-60N,收集活化后HL-60 N上清,作用于胃癌细胞,探究胃癌细胞生物学功能变化。Transwell迁移和基质胶侵袭实验检测胃癌细胞迁移、侵袭功能变化情况;MTT实验检测胃癌细胞存活情况;平板克隆实验检测胃癌细胞增殖情况。(3)采用实时荧光定量R T-PCR检测胃癌细胞共培养组和胃癌细胞条件培养基活化组HL-60N中炎症因子IL-1β、IL-6、IL-8和TNF-α等基因表达情况;实时荧光定量R T-PC R检测HL-60N共培养和活化HL-60N培养上清处理胃癌细胞EMT相关指标E-cadheri n、Vimentin、ZEB-1、S lug与干性相关指标Sox2、Oct 4和Nanog的基因表达情况;Western blot检测胃癌细胞ERK信号通路活化情况;采用ERK信号通路抑制剂预处理胃癌细胞后,再用活化HL-60N培养上清处理,Transwell实验检测胃癌细胞迁移、侵袭功能变化情况,实时荧光定量RT-PC R检测胃癌细胞EMT相关指标基因表达情况。(4)流式分选小鼠骨髓中性粒细胞,并用小鼠来源胃癌细胞MFC条件培养基处理,实时荧光定量R T-PCR检测中性粒细胞IL-1β、IL-6和TNF-α基因表达情况;ERK信号通路抑制剂预处理小鼠胃癌细胞后,再用活化小鼠骨髓中性粒细胞培养上清处理,Transwell迁移实验和基质胶侵袭实验检测小鼠胃癌细胞迁移、侵袭功能变化情况。结果:(1)与HL-60N细胞共培养后,胃癌细胞迁移、侵袭能力增强,而增殖能力无明显变化。(2)胃癌细胞条件培养基活化的HL-60N培养上清促进胃癌细胞迁移和侵袭,而对胃癌细胞增殖无显著影响。(3)HL-60N与胃癌细胞共培养或经胃癌细胞条件培养基处理后,炎症因子IL-1β、IL-6、IL-8和TNF-α基因表达上调。与HL-60N共培养或活化HL-60N培养上清处理的胃癌细胞EMT指标E-cadhe rin表达下调,Vimentin、ZEB-1和Slug表达上调,干性相关指标Sox2、Oct4和Nanog表达也上调。活化HL-60N培养上清可激活胃癌细胞ERK信号通路,阻断ERK信号通路显著抑制活化HL-60N培养上清促进胃癌细胞迁移和侵袭作用。(4)经小鼠胃癌细胞条件培养基活化的小鼠骨髓中性粒细胞IL-1β、IL-6和TNF-α基因表达上调,其培养上清促进胃癌细胞迁移和侵袭,而阻断ER K信号通路显著抑制其作用。结论:中性粒细胞与胃癌细胞共培养或经胃癌细胞条件培养基活化后,高表达炎症因子,并通过活化ERK信号通路,诱导胃癌细胞发生EMT,进而促进其迁移和侵袭。中性粒细胞与胃癌细胞的相互作用可能是其促进胃癌发生发展的重要机制,可能成为胃癌治疗的新靶点。
[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.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

【参考文献】