本文選題：小膠質(zhì)細(xì)胞 + 巨噬細(xì)胞�。� 參考：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：膠質(zhì)瘤是最常見(jiàn)的原發(fā)性中樞神經(jīng)系統(tǒng)惡性腫瘤,盡管多種治療方法均報(bào)道取得一定進(jìn)展,但總體療效改善不明顯,死亡率較高,是神經(jīng)外科領(lǐng)域的一個(gè)重要研究方向。膠質(zhì)瘤中有大量的免疫細(xì)胞浸潤(rùn),在不同的文獻(xiàn)中,這些細(xì)胞有的被稱為小膠質(zhì)細(xì)胞,有的被稱為巨噬細(xì)胞,也有的被稱為膠質(zhì)瘤相關(guān)性小膠質(zhì)/巨噬細(xì)胞(Glioma associated microglia/macrophages,GAMs)。結(jié)合文獻(xiàn)內(nèi)容,這三個(gè)名稱所指的是同一組細(xì)胞群。在膠質(zhì)瘤的生長(zhǎng)中,腦組織中特有的巨噬細(xì)胞,即小膠質(zhì)細(xì)胞被腫瘤釋放的多種趨化因子招募,浸潤(rùn)到腫瘤中心和周圍;同時(shí)由于血腦屏障的破壞,外周的巨噬細(xì)胞也浸潤(rùn)其中,和小膠質(zhì)細(xì)胞一起,共同組成了GAMs。相關(guān)研究結(jié)果提示盡管膠質(zhì)瘤組織中伴隨著大量GAMs浸潤(rùn),但在腫瘤組織中GAMs的抗腫瘤作用被抑制,其在腫瘤微環(huán)境中被馴化,反而為膠質(zhì)瘤的生長(zhǎng)、侵襲創(chuàng)造了有利條件。在膠質(zhì)瘤細(xì)胞與GAMs相互作用過(guò)程中,GAMs產(chǎn)生的IL-10、IL-18、MMP-9等細(xì)胞因子在抑制免疫、促進(jìn)腫瘤遷移過(guò)程中發(fā)揮了作用。同時(shí),膠質(zhì)瘤細(xì)胞產(chǎn)生的TGF-β、FAS配體等因子也在誘導(dǎo)、馴化GAMs使其由抗腫瘤作用向促腫瘤作用轉(zhuǎn)變。由于組成GAMs的小膠質(zhì)細(xì)胞和外周血來(lái)源的巨噬細(xì)胞均屬于巨噬細(xì)胞群,是巨噬細(xì)胞在不同時(shí)期、不同組織的特異性分化,且分子標(biāo)志物也基本一致,使膠質(zhì)瘤中這兩種細(xì)胞難以區(qū)分。這兩種具有同源性的不同來(lái)源的小膠質(zhì)細(xì)胞/巨噬細(xì)胞在膠質(zhì)瘤中生物學(xué)效應(yīng)是否一致,是一個(gè)值得探討的問(wèn)題。本研究采用體外分離培養(yǎng)小膠質(zhì)細(xì)胞及外周血巨噬細(xì)胞,然后將小膠質(zhì)細(xì)胞、巨噬細(xì)胞與C6膠質(zhì)瘤細(xì)胞株共培養(yǎng),于共培養(yǎng)0h、24h、48h行劃痕實(shí)驗(yàn),以成纖維細(xì)胞與膠質(zhì)瘤細(xì)胞共培養(yǎng)為陽(yáng)性對(duì)照,單純膠質(zhì)瘤細(xì)胞培養(yǎng)為陰性對(duì)照,每次劃痕后6h觀察實(shí)驗(yàn)結(jié)果并計(jì)算愈合率。同時(shí),于共培養(yǎng)24h、48h流式細(xì)胞分選分離小膠質(zhì)細(xì)胞、巨噬細(xì)胞及與分別這兩種細(xì)胞共培養(yǎng)的C6膠質(zhì)瘤細(xì)胞,將獲得的各組細(xì)胞與未共培養(yǎng)的三種細(xì)胞分別行WB檢測(cè),了解共培養(yǎng)前后小膠質(zhì)細(xì)胞、巨噬細(xì)胞IL-10、IL-18、MMP-9表達(dá)水平的改變及相應(yīng)C6膠質(zhì)瘤細(xì)胞株TGF-β、FAS配體表達(dá)水平的改變。主要結(jié)果劃痕實(shí)驗(yàn)提示與單純C6膠質(zhì)瘤細(xì)胞劃痕實(shí)驗(yàn)相比,共培養(yǎng)初期(0h)小膠質(zhì)細(xì)胞、巨噬細(xì)胞均表現(xiàn)為抗腫瘤效果,抑制腫瘤遷移(P0.05),但共培養(yǎng)一段時(shí)間(24h、48h)后,其抗腫瘤作用消失,兩種細(xì)胞被馴化,均表現(xiàn)為促進(jìn)腫瘤遷移且促進(jìn)效果無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。在共培養(yǎng)不同時(shí)間節(jié)點(diǎn)(24h、48h)分離細(xì)胞并提取蛋白行WB檢測(cè)小膠質(zhì)細(xì)胞、巨噬細(xì)胞的IL-10、IL-18、MMP-9蛋白表達(dá)均較0h升高,且共培養(yǎng)后兩種細(xì)胞之間蛋白表達(dá)水平無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。共培養(yǎng)24h、48h時(shí)的膠質(zhì)瘤細(xì)胞TGF-β、FAS配體表達(dá)水平較共培養(yǎng)前(0h)升高且兩組膠質(zhì)瘤細(xì)胞間無(wú)統(tǒng)計(jì)學(xué)差異(P0.05),說(shuō)明共培養(yǎng)的C6細(xì)胞株受小膠質(zhì)細(xì)胞、巨噬細(xì)胞的影響也一致。結(jié)論結(jié)合劃痕實(shí)驗(yàn)和WB檢測(cè)結(jié)果,可以認(rèn)為經(jīng)過(guò)膠質(zhì)瘤細(xì)胞馴化的小膠質(zhì)細(xì)胞與巨噬細(xì)胞轉(zhuǎn)變成了相同免疫表型的GAMs,對(duì)膠質(zhì)瘤遷移的影響一致,在體外細(xì)胞學(xué)實(shí)驗(yàn)中兩種細(xì)胞可以相互替代。
[Abstract]:Glioma is the most common primary central nervous system malignant tumor. Although a variety of therapies have been reported, the overall effect is not obvious and the mortality is high. It is an important research direction in the field of Department of neurosurgery. There are a large number of immuno cell infiltration in the glioma. In different literature, these cells have been found. Called microglia, some are known as macrophages, and some are called glioma related microglia / macrophages (Glioma associated microglia/macrophages, GAMs). Combined with the literature, these three names refer to the same group of cells. In the growth of glioma, the specific macrophages in the brain tissue, that is, microglia, are microglia. A variety of chemokines released by the tumor are recruited and infiltrated into the center and surrounding of the tumor; meanwhile, due to the destruction of the blood brain barrier, the peripheral macrophages also infiltrate, and together with microglia, the results of GAMs. related research suggest that although a large number of GAMs infiltration is associated with the glioma tissue, the anti swelling of GAMs in the tumor tissues. The tumor is inhibited, and it is domesticated in the microenvironment of the tumor. Instead, it is the growth of glioma, and the invasion creates favorable conditions. In the process of interaction between glioma cells and GAMs, the IL-10, IL-18, MMP-9 and other cytokines produced by GAMs play a role in inhibiting immunity and promoting tumor migration. At the same time, the TGF- beta, F produced by glioma cells. AS ligand and other factors are also induced, and the domestication of GAMs makes it change from anti-tumor to tumor promoting. As GAMs microglia and peripheral blood macrophages belong to the macrophage group, it is the specific differentiation of macrophages in different periods and different tissues, and the molecular markers are also basically consistent, making these two gliomas The biological effects of the two homologous microglia / macrophages in gliomas are unanimous. This study is a problem worthy of discussion. In this study, microglia and peripheral blood macrophages were isolated and cultured in vitro, and then microglia, macrophages and C6 glioma cell lines were used. Co culture, in co culture 0h, 24h, 48h line scratching experiment, the fibroblasts and glioma cells were co cultured as positive control, the simple glioma cell culture was negative control. After each scratch, 6h observed the results and calculated the healing rate. At the same time, the co culture of 24h, 48h flow cell separation and separation of microglia cells, macrophages and respectively this Two kinds of cell co cultured C6 glioma cells were detected by WB. The changes of microglia, macrophage IL-10, IL-18, MMP-9 expression level before and after co culture and the changes of the expression level of the corresponding C6 glioma cell line TGF- beta and FAS coordination were observed before and after co culture. Compared with the scratch test of simple C6 glioma cells, the microglia in the early co culture (0h) microglia showed anti tumor effect and inhibition of tumor migration (P0.05), but after a period of time (24h, 48h), the antitumor effect disappeared and two kinds of cells were domesticated, all showed no statistical difference in promoting tumor migration and promoting effect (P0.05 The expression of IL-10, IL-18 and MMP-9 protein in macrophages was higher than that of 0h in 24h, 48h and WB, and there was no significant difference in protein expression level between the two cells after co culture (P0.05). The expression level of FAS ligands was higher than that of 48h. There was no statistical difference between the two groups of glioma cells (P0.05) before co culture and two groups of glioma cells (P0.05), indicating that the co cultured C6 cell lines were affected by microglia and macrophages. Conclusion the microglia cells domesticated by glioma cells and macrophages can be transformed into the same immunophenotype with the results of scratch test and WB detection. GAMs has the same effect on glioma migration. In vitro cytology experiments, two kinds of cells can replace each other.
【學(xué)位授予單位】：第三軍醫(yī)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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