共培养体系下巨噬细胞对胃癌干细胞影响的研究

发布时间：2018-04-11 14:52

本文选题：胃癌 + 干细胞　；参考：《大连医科大学》2016年博士论文

【摘要】：背景:乳斑区聚集大量巨噬细胞,具有重要的免疫功能,参与肿瘤的发生发展、侵袭和转移,尤其在肿瘤血管生成方面起着关键作用。胃癌腹膜转移复发与胃癌细胞选择性侵袭乳斑密切相关。以往的研究发现胃癌细胞侵袭乳斑后胃癌细胞大部分被巨噬细胞消灭。但残留在乳斑内一小部分细胞可以在巨噬细胞环境中继续生存并出现扩增。考虑这一部分具有复制能力和免疫逃逸的残留细胞为胃癌干细胞。目的:为研究乳斑区巨噬细胞对胃癌干细胞的作用影响,本实验设计了胃癌干细胞与巨噬细胞共培养体系,在共培养体系下模拟乳斑区胃癌干细胞的微环境,拟探求胃癌干细胞微环境的变化。方法:实验首先通过成球法从人胃癌细胞系SGC-7901细胞培养、分离、传代胃癌干细胞,通过荧光染料Hoechst33342对细胞进行染色,应用流式细胞仪监测并分选胃癌干细胞,为共培养实验提供胃癌干细胞。在建立的共培养体系下,用MTT法检测胃癌干细胞存活率。监测胃癌干细胞平板克隆能力的变化以及应用RT-PCR法、Western Blot、ELASA法检测胃癌干细胞表达Caspase-3/9、IL-4,IL-10、INF-γ、IL-12、MMP-2、MMP-9、MCP-1、COX-2、PGE2、VEGF、TGF-β的变化。结果:共培养体系下胃癌干细胞活力随巨噬细胞浓度的升高2×105/ml、4×105/ml后被明显抑制。共培养体系下无巨噬细胞组的胃癌干细胞克隆形成率为(0.811±0.035)%,1×105细胞组胃癌干细胞的克隆形成率为(0.612±0.157)%,2×105细胞组的胃癌干细胞克隆形成率为(0.4057±0.016)%,4×105细胞组的胃癌干细胞克隆形成率为(0.213±0.413)%,差异有统计学意义(P0.01)。共培养体系下胃癌干细胞表达Caspase-3/9水平随着巨噬细胞浓度的升高而升高,表现出胃癌干细胞凋亡的激活。MCP-1是最主要的趋化因子,对肿瘤的生长、侵袭以及新生血管的生成有重要作用,在巨噬细胞共培养下,胃癌干细胞表现出MCP-1活性增强,提示在炎性环境下干细胞有被激活的可能。COX-2是花生四烯酸代谢的关键限速酶,具有多种抗肿瘤凋亡途径,可以降低介导凋亡的转化生长因子TGF-β的水平,限制细胞凋亡,有促进肿瘤生长的作用。共培养体系下胃癌干细胞高表达Co X-2,随巨噬细胞浓度升高表现出更高的抗凋亡能力。于巨噬细胞共培养体系下胃癌干细胞低表达COX-2下游的TGF-β,在炎性环境下抑制了TGF-βm RNA及蛋白的表达。共培养体系下随巨噬细胞浓度升高,胃癌干细胞高表达PGE2 m RNA及蛋白活性,抑制干细胞的凋亡,促进周围血管生成。共培养体系下胃癌干细胞VEGF的m RNA及蛋白的表达被1×105、2×105/ml、4×105/ml的巨噬细胞显著激活。肿瘤细胞和肿瘤相关巨噬细胞都可以分泌免疫抑制因子IL-10,IL-10可以促进巨噬细胞分化成M2型肿瘤相关巨噬细胞,抑制炎症反应、促进血管生成和组织修复。在与巨噬细胞共培养环境下胃癌干细胞表达IL-10活性减低。IL-4在胃癌患者血清中高表达,IL-4与IL-10同样在M2型巨噬细胞高表达,胃癌干细胞在与巨噬细胞共培养环境下低表达IL-4。IL-12对肿瘤的生长和转移具有抑制作用,M1型巨噬细胞高表达IL-12,在于巨噬细胞共培养环境下胃癌干细胞高表达IL-12。在与巨噬细胞共培养环境下,胃癌干细胞虽然表达IL-4、IL-10与表达IL-12呈现双向性,但总体反映了胃癌干细胞活性受到巨噬细胞的抑制。胃癌干细胞在巨噬细胞环境下高表达IFN-γ,表现出干细胞活性受到巨噬细胞抑制。MMP-2和MMP-9属基质金属蛋白酶,能够影响细胞黏附分子,介导肿瘤细胞对宿主细胞的细胞外基质降解,调控肿瘤新生血管的生成。胃癌干细胞在巨噬细胞影响低表达MMP-2和MMP-9,干细胞的侵袭性受到巨噬细胞抑制。结论:胃癌干细胞与巨噬细胞共培养体系下,虽然MTT实验和干细胞克隆实验中表现为胃癌干细胞受到巨噬细胞的抑制,共培养体系中Caspase-3/9、IL-4,IL-10、INF-γ受到激活、IL-12、MMP-2、MMP-9基质金属蛋白酶受到抑制;同时也高表达了MCP-1、COX-2、PGE2、VEGF、低表达TGF-β,反映出胃癌干细胞在受到免疫抑制的同时仍具备抗凋亡、促进血管新生的能力。纵观胃癌干细胞与巨噬细胞共培养体系,胃癌干细胞在免疫抑制的影响下表现出了细胞数量的减少和克隆能力降低。胃癌的免疫治疗仍是抗肿瘤治疗的研究方向。
[Abstract]:Background: the Milky spot gathered a large number of macrophages, has important immune functions, is involved in tumor development, invasion and metastasis, especially in tumor angiogenesis plays a key role. Peritoneal metastasis of gastric cancer recurrence and selective gastric cancer cells invasion and milky spots are closely related. Previous studies have found that the invasion of the milky spots in gastric cancer cells after gastric cancer cells are most macrophage destroyed. In the milky spots but remained in a small number of cells can survive in the environment and the emergence of macrophage amplification. Consider this part has residual cell replication ability and immune escape of gastric cancer stem cells. Objective: To study the role of macrophage in milky spot of gastric cancer stem cells of the experimental design co cultivation system of gastric cancer stem cells and macrophages in the microenvironment of gastric cancer, simulation of the Milky spot stem cell co culture system, to explore the gastric cancer stem cell microenvironment change Methods: the experiment. First, through the ball forming method from SGC-7901 cell culture, human gastric cancer cell lines were isolated, gastric cancer stem cells, the cells by fluorescent dye Hoechst33342 staining, flow cytometry was used to monitor and sorting of gastric cancer stem cells, the gastric cancer stem cells were co cultured in the co culture system established under the detection of gastric cancer stem cell survival rate by MTT method. Monitoring the changes of gastric cancer stem cell clone ability and the application of RT-PCR, Western Blot, ELASA method for detection of gastric cancer stem cells express Caspase-3/9, IL-4, IL-10, IL-12, MMP-2, INF-, MMP-9, MCP-1, COX-2, PGE2, VEGF, TGF-. Results: the changes of beta under the system of gastric cancer stem cell activity increased with the concentration of 2 * 105/ml macrophage co culture, 4 * 105/ml was significantly inhibited. No macrophage system group of gastric cancer stem cell clone formation rate of co culture (0.811 + 0.035)%, 1 * 105 cells of stomach Cancer stem cell clone formation rate was (0.612 + 0.157)%, 2 * 105 cell groups of gastric cancer stem cell clone formation rate was (0.4057 + 0.016)%, 4 * 105 cell groups of gastric cancer stem cell clone formation rate was (0.213 + 0.413)%, the difference was statistically significant (P0.01) system. Under the gastric cancer stem cells the expression level of Caspase-3/9 increased with the concentration of macrophages were cultured in vitro and showed activation of.MCP-1 cell apoptosis of gastric cancer stem is the main chemotactic factor, on tumor growth, invasion and angiogenesis plays an important role in macrophages co cultured, gastric cancer stem cells showed enhanced MCP-1 activity in the inflammatory environment, suggesting that stem cells may be the activation of.COX-2 is a key rate limiting enzyme four arachidonic acid metabolism, with a variety of anti - tumor apoptosis pathway mediated apoptosis can reduce the transforming growth factor TGF- beta level limit cell apoptosis, promote The tumor growth. Under the system of gastric cancer stem cells with high expression of Co in X-2 co culture showed higher anti apoptosis ability with macrophage concentration. In macrophages co cultured with gastric cancer stem cells in the low expression of COX-2 downstream of the TGF- beta, the expression of TGF- beta m RNA and protein inhibition in inflammatory conditions. System with elevated macrophage co culture of gastric cancer stem cells with high expression of PGE2 M protein and RNA activity, inhibit stem cell apoptosis, promote peripheral angiogenesis. Expression of M co culture system of RNA and protein in gastric cancer stem cell VEGF is 1 * 105,2 * 105/ml, 4 * 105/ml macrophages significantly activated tumor cells. And tumor associated macrophages can secrete immunosuppressive factor IL-10, IL-10 can promote the differentiation of macrophages into type M2 tumor associated macrophages, inhibiting the inflammatory reaction, promote angiogenesis and tissue repair in macrophage and. Under the environment of gastric cancer stem cells expressing IL-10 activity to reduce the high expression of.IL-4 in serum of patients with gastric cancer cells, IL-4 and IL-10 are also highly expressed in M2 macrophages, cells in coculture with macrophages under the environment of low expression of IL-4.IL-12 on tumor growth and metastasis has inhibitory effect on gastric cancer stem M1 macrophages, high expression of IL-12 is. Under the environment of high expression of IL-12. in gastric cancer stem cells and macrophages were cultured under co cultured macrophages, gastric cancer stem cells while the expression of IL-4, IL-12 and IL-10 expression showed bidirectional, but generally reflects the inhibition of gastric cancer stem cell activity by macrophages. Gastric cancer stem cells in macrophages under the environment of high IFN- expression, showing stem cell activity.MMP-2 and MMP-9 by macrophage inhibition of matrix metalloproteinase, can affect cell adhesion molecules, cell mediated tumor cells to host cells Extracellular matrix degradation, generation regulation of tumor neovascularization. Cells in macrophages affected the low expression of MMP-2 and MMP-9 in gastric cancer stem cells, stem invasion by macrophage inhibition. Conclusion: the co culture system of gastric cancer stem cells and macrophages, although MTT and experimental stem cell cloning experiment showed gastric cancer stem cells by macrophage inhibition. In the co culture system of Caspase-3/9, IL-4, IL-10, INF-, IL-12, MMP-2 activation by gamma, MMP-9, matrix metalloproteinase inhibition; also the high expression of MCP-1, COX-2, PGE2, VEGF, low expression of TGF- beta, reflects the gastric cancer stem cells have still anti apoptosis simultaneously by immune suppression, the ability to promote angiogenesis the co culture system. The gastric cancer stem cells and macrophages, gastric cancer stem cells in immune suppression showed a decrease in the number of cells and cloning ability decreased. Immunotherapy of gastric cancer still It is the research direction of antitumor therapy.

【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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