本文選題：胃癌 + 干細胞�。� 參考：《大連醫(yī)科大學(xué)》2016年博士論文

【摘要】：背景:乳斑區(qū)聚集大量巨噬細胞,具有重要的免疫功能,參與腫瘤的發(fā)生發(fā)展、侵襲和轉(zhuǎn)移,尤其在腫瘤血管生成方面起著關(guān)鍵作用。胃癌腹膜轉(zhuǎn)移復(fù)發(fā)與胃癌細胞選擇性侵襲乳斑密切相關(guān)。以往的研究發(fā)現(xiàn)胃癌細胞侵襲乳斑后胃癌細胞大部分被巨噬細胞消滅。但殘留在乳斑內(nèi)一小部分細胞可以在巨噬細胞環(huán)境中繼續(xù)生存并出現(xiàn)擴增。考慮這一部分具有復(fù)制能力和免疫逃逸的殘留細胞為胃癌干細胞。目的:為研究乳斑區(qū)巨噬細胞對胃癌干細胞的作用影響,本實驗設(shè)計了胃癌干細胞與巨噬細胞共培養(yǎng)體系,在共培養(yǎng)體系下模擬乳斑區(qū)胃癌干細胞的微環(huán)境,擬探求胃癌干細胞微環(huán)境的變化。方法:實驗首先通過成球法從人胃癌細胞系SGC-7901細胞培養(yǎng)、分離、傳代胃癌干細胞,通過熒光染料Hoechst33342對細胞進行染色,應(yīng)用流式細胞儀監(jiān)測并分選胃癌干細胞,為共培養(yǎng)實驗提供胃癌干細胞。在建立的共培養(yǎng)體系下,用MTT法檢測胃癌干細胞存活率。監(jiān)測胃癌干細胞平板克隆能力的變化以及應(yīng)用RT-PCR法、Western Blot、ELASA法檢測胃癌干細胞表達Caspase-3/9、IL-4,IL-10、INF-γ、IL-12、MMP-2、MMP-9、MCP-1、COX-2、PGE2、VEGF、TGF-β的變化。結(jié)果:共培養(yǎng)體系下胃癌干細胞活力隨巨噬細胞濃度的升高2×105/ml、4×105/ml后被明顯抑制。共培養(yǎng)體系下無巨噬細胞組的胃癌干細胞克隆形成率為(0.811±0.035)%,1×105細胞組胃癌干細胞的克隆形成率為(0.612±0.157)%,2×105細胞組的胃癌干細胞克隆形成率為(0.4057±0.016)%,4×105細胞組的胃癌干細胞克隆形成率為(0.213±0.413)%,差異有統(tǒng)計學(xué)意義(P0.01)。共培養(yǎng)體系下胃癌干細胞表達Caspase-3/9水平隨著巨噬細胞濃度的升高而升高,表現(xiàn)出胃癌干細胞凋亡的激活。MCP-1是最主要的趨化因子,對腫瘤的生長、侵襲以及新生血管的生成有重要作用,在巨噬細胞共培養(yǎng)下,胃癌干細胞表現(xiàn)出MCP-1活性增強,提示在炎性環(huán)境下干細胞有被激活的可能。COX-2是花生四烯酸代謝的關(guān)鍵限速酶,具有多種抗腫瘤凋亡途徑,可以降低介導(dǎo)凋亡的轉(zhuǎn)化生長因子TGF-β的水平,限制細胞凋亡,有促進腫瘤生長的作用。共培養(yǎng)體系下胃癌干細胞高表達Co X-2,隨巨噬細胞濃度升高表現(xiàn)出更高的抗凋亡能力。于巨噬細胞共培養(yǎng)體系下胃癌干細胞低表達COX-2下游的TGF-β,在炎性環(huán)境下抑制了TGF-βm RNA及蛋白的表達。共培養(yǎng)體系下隨巨噬細胞濃度升高,胃癌干細胞高表達PGE2 m RNA及蛋白活性,抑制干細胞的凋亡,促進周圍血管生成。共培養(yǎng)體系下胃癌干細胞VEGF的m RNA及蛋白的表達被1×105、2×105/ml、4×105/ml的巨噬細胞顯著激活。腫瘤細胞和腫瘤相關(guān)巨噬細胞都可以分泌免疫抑制因子IL-10,IL-10可以促進巨噬細胞分化成M2型腫瘤相關(guān)巨噬細胞,抑制炎癥反應(yīng)、促進血管生成和組織修復(fù)。在與巨噬細胞共培養(yǎng)環(huán)境下胃癌干細胞表達IL-10活性減低。IL-4在胃癌患者血清中高表達,IL-4與IL-10同樣在M2型巨噬細胞高表達,胃癌干細胞在與巨噬細胞共培養(yǎng)環(huán)境下低表達IL-4。IL-12對腫瘤的生長和轉(zhuǎn)移具有抑制作用,M1型巨噬細胞高表達IL-12,在于巨噬細胞共培養(yǎng)環(huán)境下胃癌干細胞高表達IL-12。在與巨噬細胞共培養(yǎng)環(huán)境下,胃癌干細胞雖然表達IL-4、IL-10與表達IL-12呈現(xiàn)雙向性,但總體反映了胃癌干細胞活性受到巨噬細胞的抑制。胃癌干細胞在巨噬細胞環(huán)境下高表達IFN-γ,表現(xiàn)出干細胞活性受到巨噬細胞抑制。MMP-2和MMP-9屬基質(zhì)金屬蛋白酶,能夠影響細胞黏附分子,介導(dǎo)腫瘤細胞對宿主細胞的細胞外基質(zhì)降解,調(diào)控腫瘤新生血管的生成。胃癌干細胞在巨噬細胞影響低表達MMP-2和MMP-9,干細胞的侵襲性受到巨噬細胞抑制。結(jié)論:胃癌干細胞與巨噬細胞共培養(yǎng)體系下,雖然MTT實驗和干細胞克隆實驗中表現(xiàn)為胃癌干細胞受到巨噬細胞的抑制,共培養(yǎng)體系中Caspase-3/9、IL-4,IL-10、INF-γ受到激活、IL-12、MMP-2、MMP-9基質(zhì)金屬蛋白酶受到抑制;同時也高表達了MCP-1、COX-2、PGE2、VEGF、低表達TGF-β,反映出胃癌干細胞在受到免疫抑制的同時仍具備抗凋亡、促進血管新生的能力。縱觀胃癌干細胞與巨噬細胞共培養(yǎng)體系,胃癌干細胞在免疫抑制的影響下表現(xiàn)出了細胞數(shù)量的減少和克隆能力降低。胃癌的免疫治療仍是抗腫瘤治療的研究方向。
[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.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.2

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