本文選題：小膠質細胞 + 巨噬細胞　； 參考：《第三軍醫(yī)大學》2017年碩士論文

【摘要】：膠質瘤是最常見的原發(fā)性中樞神經系統(tǒng)惡性腫瘤,盡管多種治療方法均報道取得一定進展,但總體療效改善不明顯,死亡率較高,是神經外科領域的一個重要研究方向。膠質瘤中有大量的免疫細胞浸潤,在不同的文獻中,這些細胞有的被稱為小膠質細胞,有的被稱為巨噬細胞,也有的被稱為膠質瘤相關性小膠質/巨噬細胞(Glioma associated microglia/macrophages,GAMs)。結合文獻內容,這三個名稱所指的是同一組細胞群。在膠質瘤的生長中,腦組織中特有的巨噬細胞,即小膠質細胞被腫瘤釋放的多種趨化因子招募,浸潤到腫瘤中心和周圍;同時由于血腦屏障的破壞,外周的巨噬細胞也浸潤其中,和小膠質細胞一起,共同組成了GAMs。相關研究結果提示盡管膠質瘤組織中伴隨著大量GAMs浸潤,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,反而為膠質瘤的生長、侵襲創(chuàng)造了有利條件。在膠質瘤細胞與GAMs相互作用過程中,GAMs產生的IL-10、IL-18、MMP-9等細胞因子在抑制免疫、促進腫瘤遷移過程中發(fā)揮了作用。同時,膠質瘤細胞產生的TGF-β、FAS配體等因子也在誘導、馴化GAMs使其由抗腫瘤作用向促腫瘤作用轉變。由于組成GAMs的小膠質細胞和外周血來源的巨噬細胞均屬于巨噬細胞群,是巨噬細胞在不同時期、不同組織的特異性分化,且分子標志物也基本一致,使膠質瘤中這兩種細胞難以區(qū)分。這兩種具有同源性的不同來源的小膠質細胞/巨噬細胞在膠質瘤中生物學效應是否一致,是一個值得探討的問題。本研究采用體外分離培養(yǎng)小膠質細胞及外周血巨噬細胞,然后將小膠質細胞、巨噬細胞與C6膠質瘤細胞株共培養(yǎng),于共培養(yǎng)0h、24h、48h行劃痕實驗,以成纖維細胞與膠質瘤細胞共培養(yǎng)為陽性對照,單純膠質瘤細胞培養(yǎng)為陰性對照,每次劃痕后6h觀察實驗結果并計算愈合率。同時,于共培養(yǎng)24h、48h流式細胞分選分離小膠質細胞、巨噬細胞及與分別這兩種細胞共培養(yǎng)的C6膠質瘤細胞,將獲得的各組細胞與未共培養(yǎng)的三種細胞分別行WB檢測,了解共培養(yǎng)前后小膠質細胞、巨噬細胞IL-10、IL-18、MMP-9表達水平的改變及相應C6膠質瘤細胞株TGF-β、FAS配體表達水平的改變。主要結果劃痕實驗提示與單純C6膠質瘤細胞劃痕實驗相比,共培養(yǎng)初期(0h)小膠質細胞、巨噬細胞均表現(xiàn)為抗腫瘤效果,抑制腫瘤遷移(P0.05),但共培養(yǎng)一段時間(24h、48h)后,其抗腫瘤作用消失,兩種細胞被馴化,均表現(xiàn)為促進腫瘤遷移且促進效果無統(tǒng)計學差異(P0.05)。在共培養(yǎng)不同時間節(jié)點(24h、48h)分離細胞并提取蛋白行WB檢測小膠質細胞、巨噬細胞的IL-10、IL-18、MMP-9蛋白表達均較0h升高,且共培養(yǎng)后兩種細胞之間蛋白表達水平無統(tǒng)計學差異(P0.05)。共培養(yǎng)24h、48h時的膠質瘤細胞TGF-β、FAS配體表達水平較共培養(yǎng)前(0h)升高且兩組膠質瘤細胞間無統(tǒng)計學差異(P0.05),說明共培養(yǎng)的C6細胞株受小膠質細胞、巨噬細胞的影響也一致。結論結合劃痕實驗和WB檢測結果,可以認為經過膠質瘤細胞馴化的小膠質細胞與巨噬細胞轉變成了相同免疫表型的GAMs,對膠質瘤遷移的影響一致,在體外細胞學實驗中兩種細胞可以相互替代。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.41

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