大鼠小胶质细胞及巨噬细胞对C6胶质瘤细胞株迁移能力影响的研究

发布时间：2018-06-10 13:18

本文选题：小胶质细胞 + 巨噬细胞　；参考：《第三军医大学》2017年硕士论文

【摘要】：胶质瘤是最常见的原发性中枢神经系统恶性肿瘤,尽管多种治疗方法均报道取得一定进展,但总体疗效改善不明显,死亡率较高,是神经外科领域的一个重要研究方向。胶质瘤中有大量的免疫细胞浸润,在不同的文献中,这些细胞有的被称为小胶质细胞,有的被称为巨噬细胞,也有的被称为胶质瘤相关性小胶质/巨噬细胞(Glioma associated microglia/macrophages,GAMs)。结合文献内容,这三个名称所指的是同一组细胞群。在胶质瘤的生长中,脑组织中特有的巨噬细胞,即小胶质细胞被肿瘤释放的多种趋化因子招募,浸润到肿瘤中心和周围;同时由于血脑屏障的破坏,外周的巨噬细胞也浸润其中,和小胶质细胞一起,共同组成了GAMs。相关研究结果提示尽管胶质瘤组织中伴随着大量GAMs浸润,但在肿瘤组织中GAMs的抗肿瘤作用被抑制,其在肿瘤微环境中被驯化,反而为胶质瘤的生长、侵袭创造了有利条件。在胶质瘤细胞与GAMs相互作用过程中,GAMs产生的IL-10、IL-18、MMP-9等细胞因子在抑制免疫、促进肿瘤迁移过程中发挥了作用。同时,胶质瘤细胞产生的TGF-β、FAS配体等因子也在诱导、驯化GAMs使其由抗肿瘤作用向促肿瘤作用转变。由于组成GAMs的小胶质细胞和外周血来源的巨噬细胞均属于巨噬细胞群,是巨噬细胞在不同时期、不同组织的特异性分化,且分子标志物也基本一致,使胶质瘤中这两种细胞难以区分。这两种具有同源性的不同来源的小胶质细胞/巨噬细胞在胶质瘤中生物学效应是否一致,是一个值得探讨的问题。本研究采用体外分离培养小胶质细胞及外周血巨噬细胞,然后将小胶质细胞、巨噬细胞与C6胶质瘤细胞株共培养,于共培养0h、24h、48h行划痕实验,以成纤维细胞与胶质瘤细胞共培养为阳性对照,单纯胶质瘤细胞培养为阴性对照,每次划痕后6h观察实验结果并计算愈合率。同时,于共培养24h、48h流式细胞分选分离小胶质细胞、巨噬细胞及与分别这两种细胞共培养的C6胶质瘤细胞,将获得的各组细胞与未共培养的三种细胞分别行WB检测,了解共培养前后小胶质细胞、巨噬细胞IL-10、IL-18、MMP-9表达水平的改变及相应C6胶质瘤细胞株TGF-β、FAS配体表达水平的改变。主要结果划痕实验提示与单纯C6胶质瘤细胞划痕实验相比,共培养初期(0h)小胶质细胞、巨噬细胞均表现为抗肿瘤效果,抑制肿瘤迁移(P0.05),但共培养一段时间(24h、48h)后,其抗肿瘤作用消失,两种细胞被驯化,均表现为促进肿瘤迁移且促进效果无统计学差异(P0.05)。在共培养不同时间节点(24h、48h)分离细胞并提取蛋白行WB检测小胶质细胞、巨噬细胞的IL-10、IL-18、MMP-9蛋白表达均较0h升高,且共培养后两种细胞之间蛋白表达水平无统计学差异(P0.05)。共培养24h、48h时的胶质瘤细胞TGF-β、FAS配体表达水平较共培养前(0h)升高且两组胶质瘤细胞间无统计学差异(P0.05),说明共培养的C6细胞株受小胶质细胞、巨噬细胞的影响也一致。结论结合划痕实验和WB检测结果,可以认为经过胶质瘤细胞驯化的小胶质细胞与巨噬细胞转变成了相同免疫表型的GAMs,对胶质瘤迁移的影响一致,在体外细胞学实验中两种细胞可以相互替代。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

【参考文献】