M2样肿瘤相关巨噬细胞对胶质瘤血管拟态的影响及机制研究

发布时间：2018-05-06 19:38

本文选题：胶质瘤 + 血管拟态　；参考：《山东大学》2017年博士论文

【摘要】：研究背景脑胶质瘤(Glioma)是中枢神经系统中最常见的颅内原发性恶性肿瘤,约占脑内原发肿瘤的50%,其中以高度恶性的胶质母细胞瘤(Glioblastomamultiforme,GBM;WHO Ⅳ级)最为常见。近几年来,虽然临床上手术联合放化疗等综合治疗手段,在一定程度上改善了恶性胶质瘤的治疗疗效,但胶质母细胞瘤患者的中位总生存期仍然仅有15-18个月,具有预后差、复发率高的特点。由于胶质瘤是一种高度血管化的脑肿瘤,因此,靶向血管内皮细胞的抗血管生成药物治疗胶质瘤的应用前景备受瞩目。然而,近期的数据发现,这种看似非常具有潜力的抗血管疗法,临床应用后却出现了疗效短暂和疗效欠佳的问题。直到1999年血管拟态(Vasculogenic Mimicry，，VM)概念的提出,人们才意识到在肿瘤组织中除了由内皮细胞构成的血管外,还有其他的血液供应形式。血管拟态是由肿瘤细胞在特定情况下模拟血管内皮细胞形成的一种功能上类似于正常血管的管道,其内可含有红细胞,并且可以与常规的内皮血管相通连,构成网状的血液输送循环,从而为肿瘤提供必要的营养支持。目前已经证实,包括胶质瘤在内的多种恶性肿瘤中均存在血管拟态,并且其阳性率与肿瘤级别及患者的不良预后密切相关。因而,靶向血管拟态的治疗策略很可能是一种非常有前景的抗肿瘤血管治疗手段,所以,在对胶质瘤患者进行传统抗血管治疗的同时,联合应用抑制肿瘤血管拟态形成的药物可能会有更好的治疗疗效。已经有越来越多的研究表明,肿瘤微环境在血管拟态形成过程中具有不可或缺的作用,但具体分子机制尚未明确,深入研究肿瘤微环境将会促进人们对血管拟态相关机制的了解。免疫细胞浸润是多种肿瘤微环境的一个显著特征,在肿瘤的侵袭、血管新生以及转移等恶性生物学行为中发挥着关键作用,进而影响了肿瘤的进展。在这群免疫细胞中巨噬细胞的浸润比率最高,研究发现,胶质母细胞瘤中浸润的肿瘤相关巨噬细胞比例高达30%,且高表达CD14、CD68、CD163和CD204等M2型巨噬细胞标志物。既往研究发现,肿瘤局部浸润巨噬细胞的数量及活性与患者的预后密切相关。此外,有报道证实,在既往接受抗血管治疗后病情复发的胶质母细胞瘤患者中,瘤体内浸润巨噬细胞的数量明显增加,并且与肿瘤的血管密度呈正相关。这些结果表明,在肿瘤进展过程中,肿瘤浸润巨噬细胞对肿瘤血供的形成发挥着不容忽视的作用。因此,靶向肿瘤浸润巨噬细胞的促血管生成作用,也许是克服胶质瘤抗血管药物抵抗的有效途径。肿瘤微环境的另一个重要特征是其内存在大量独特的免疫介质,包括细胞因子以及趋化因子。越来越多的数据表明,这类免疫介质可以通过调节免疫细胞和肿瘤细胞的功能,营造出一个有助于肿瘤进展的微环境。白细胞介素-6(Interleukin-6,IL-6)就是这样一种功能多效的免疫介质,其广泛存在于多种恶性实体瘤的炎性微环境中,通过参与免疫调节、促进肿瘤细胞增殖、迁移、存活以及药物耐药,引发肿瘤患者的不良预后。此外,还有结果显示,IL-6与肿瘤抗血管治疗效果不佳有着密不可分的关系。在胶质瘤中,IL-6可以通过增强血管内皮细胞的迁移来促进肿瘤的侵袭和血管生成,阻断IL-6同时合用贝伐单抗可以更有效的阻碍胶质瘤的侵袭和生长。这些结果表明,肿瘤微环境中IL-6过表达会明显影响肿瘤的血管生成,但IL-6是否在胶质瘤血管拟态形成过程中同样扮演着重要的角色,目前仍不清楚。本课题拟在已有研究的基础上,利用免疫组化的方法初步探索血管拟态与肿瘤相关巨噬细胞在人胶质瘤组织中的表达特点、临床意义及二者的相关性。通过体外实验,分析THP-1衍生M2巨噬细胞和外周血单核衍生M2巨噬细胞是否会影响胶质瘤血管拟态的形成,并探讨其相关的作用机制。第一部分血管拟态和CD163+巨噬细胞在胶质瘤中的表达目的检测血管拟态和CD163+巨噬细胞在胶质瘤组织中的表达情况,并初步分析两者之间是否存在相关性,以及其与患者预后和临床病理因素之间的关系。方法1.本研究回顾性纳入山东大学齐鲁医院行胶质瘤切除术的患者87例,获取肿瘤组织样本。采用免疫组织化学方法检测血管拟态和CD163+巨噬细胞在胶质瘤中的表达情况,并分析两者之间的相关性。2.收集患者的基本信息,包括性别、年龄等基本临床资料,分析血管拟态和CD163+巨噬细胞与胶质瘤患者临床病理因素及预后的关系。结果1.通过免疫组化对胶质瘤组织样本的连续切片进行CD31/PAS和CD163染色发现,高级别胶质瘤(WHO分级Ⅲ-Ⅳ级)的血管拟态(CD31-/PAS+)水平和CD163表达密度明显高于低级别的(WHO分级Ⅰ-Ⅱ级)(P0.01,P0.01)。相关性分析结果显示,血管拟态水平与CD163表达密度在胶质瘤组织中呈正相关(r2=0.416,P0.001)。2.分析胶质瘤组织中血管拟态和CD163的表达与不同病理因素之间的关系发现,二者均与肿瘤的级别有关(P0.001,P0.001),血管拟态还与肿瘤的大小有关(P=0.027);二者与性别、年龄、肿瘤位置、KPS评分以及肿瘤的生长方式无关。3.Kaplan-Meier分析发现,与CD163表达水平较高的胶质瘤患者相比,CD163表达水平较低的胶质瘤患者具有显著的生存优势(p=0.006);血管拟态低水平的胶质瘤患者其中位生存时间明显长于血管拟态高水平的胶质瘤患者(P0.001)。进一步分析发现,CD163高表达同时血管拟态高水平胶质瘤患者的中位生存时间明显短于其他组的胶质瘤患者(P0.001)。结论1.血管拟态水平和CD163+巨噬细胞浸润数量均与胶质瘤级别有关,二者在胶质瘤组织中的表达呈正相关。2.CD163+巨噬细胞浸润和血管拟态存在与胶质瘤患者的不良预后有关。3.血管拟态水平和CD163+巨噬细胞浸润数量与胶质瘤患者的临床病理特征,如性别、年龄、肿瘤位置、KPS评分以及肿瘤的生长方式等无关。第二部分THP-1衍生M2巨噬细胞调控胶质瘤血管拟态及机制研究目的探索THP-1衍生M2巨噬细胞是否影响胶质瘤血管拟态的形成及其可能的机制方法1.采用qRT-PCR方法检测THP-1衍生巨噬细胞的表型。2.利用成管实验检测THP-1衍生M2巨噬细胞对胶质瘤细胞血管拟态形成的影响。3.采用qRT-PCR、Western blotting等方法检测THP-1衍生M2巨噬细胞对胶质瘤细胞血管拟态相关标志物转录和蛋白水平的影响。4.采用悬浮芯片和ELISA等技术检测THP-1衍生M2巨噬细胞对胶质瘤细胞IL-6分泌的影响。5.利用qRT-PCR实验技术检测THP-1衍生M2巨噬细胞对胶质瘤细胞IL-6和血管拟态相关指标表达时序的影响。6.采用成管实验、Western blotting等方法检测IL-6是否影响胶质瘤血管拟态的形成。7.利用成管实验、qRT-PCR、ELISA及Western blotting等方法探索THP-1衍生M2巨噬细胞对胶质瘤细胞相关信号机制的影响。结果1.THP-1衍生M2巨噬细胞表达高水平的M2型标记物(CD163,CD206,IL-10,IL-1RA 和 CCL17)和低水平的 M1 型标记物(TNF-α,IL-1β 和 IL-12)。2.与THP-1上清处理组或对照组相比,THP-1衍生M2巨噬细胞的上清明显促进胶质瘤细胞血管拟态的形成(U251,P0.001;A172,P0.01)和血管拟态相关指标(MMP-9,MMP-14,LAMC2)的表达。3.THP-1衍生M2巨噬细胞上清促进胶质瘤细胞IL-6的分泌,并呈现时间和浓度依赖的特点,且IL-6表达上调时间(约6h)要早于血管拟态相关指标表达升高的时间(约12h)。4.IL-6中和抗体(1 μg/ml)可以阻断THP-1衍生M2巨噬细胞诱导的胶质瘤细胞血管拟态形成(U251,P0.01;A172,P0.01)和相关指标表达的上调。此外,重组人IL-6(rhIL-6,100ng/ml)能够促进胶质瘤细胞血管拟态的形成(U251,P0.01;A172,P0.05)和相关指标的表达。5.THP-1衍生M2巨噬细胞上清升高胶质瘤细胞PKC信号通路的磷酸化水平。PKC抑制剂(Bisindolylmaleimide I)可以抑制THP-1衍生M2巨噬细胞诱导的胶质瘤细胞IL-6的转录(U251,P0.01;A172,P0.01)和分泌(U251,P0.001;A172,P0.001)水平的上调,进而抑制胶质瘤细胞血管拟态的形成(U251,P0.05;A172,P0.05)和相关指标的表达。结论1.THP-1衍生M2巨噬细胞体外促进胶质瘤细胞血管拟态的形成。2.THP-1衍生M2巨噬细胞通过激活胶质瘤细胞PKC信号通路促进IL-6的产生,进而促进血管拟态的形成。第三部分单核细胞衍生M2巨噬细胞调控胶质瘤血管拟态及机制研究目的探索人外周血单核细胞衍生M2巨噬细胞是否影响胶质瘤细胞血管拟态的形成及其作用机制方法1.利用磁珠分选法从人外周血分离CD14+单核细胞,并用相关诱导因子将其培养成巨噬细胞。2.采用qRT-PCR和ELISA技术检测人外周血单核细胞衍生巨噬细胞的表型。3.利用qRT-PCR和ELISA实验方法分析人外周血单核细胞衍生M2巨噬细胞对胶质瘤细胞IL-6分泌的影响。4.采用成管实验检测人外周血单核细胞衍生M2巨噬细胞对胶质瘤细胞血管拟态形成的影响。5.采用成管实验检测胶质瘤细胞分泌的IL-6对血管拟态形成的影响。结果1.人外周血单核细胞衍生M2巨噬细胞表达高转录水平的M2型标记物(CD163,CD206,CCL18和CCL17)和低转录水平的M1型标记物(TNF-α,IL-1β,IL-6和IL-12)。单核细胞衍生M2巨噬细胞分泌高水平的M2型标记物(CCL22,CCL18,CCL17 和 IL-10)和低水平的 M1 型标记物(IL1-β,IL-6,IL-12 和 IL-8)。2.人外周血单核细胞衍生M2巨噬细胞的上清上调胶质瘤细胞IL-6的转录(U251,P0.01;A172,P0.001)和分泌(U251,P0.001;A172,P0.001)水平。3.将IL-6中和抗体(1μg/ml)加入共培养体系可以显著降低胶质瘤细胞管状结构的形成数量(U251,P0.01;A172,P0.01)。这表明IL-6中和抗体可以阻断人外周血单核细胞衍生M2巨噬细胞介导的胶质瘤细胞血管拟态形成的增强作用。结论1.人外周血单核细胞衍生M2巨噬细胞升高胶质瘤细胞IL-6的表达。2.人外周血单核细胞衍生M2巨噬细胞通过增加胶质瘤细胞IL-6的分泌,进一步促进胶质瘤血管拟态的形成。
[Abstract]:Background brain glioma (Glioma) is the most common primary intracranial malignant tumor in the central nervous system, accounting for about 50% of the primary brain tumors, and highly malignant glioblastoma (Glioblastomamultiforme, GBM; WHO IV) is the most common. The therapeutic effect of malignant glioma is improved to a certain extent, but the median survival period of the patients with glioblastoma is still only 15-18 months, with poor prognosis and high recurrence rate. As glioma is a highly vascularized brain tumor, the application prospect of antiangiogenic drugs targeting vascular endothelial cells in the treatment of glioma Recent data, however, have found that this seemingly very promising antiangio therapy has a short and poor curative effect after clinical applications. It was not until 1999 that the concept of Vasculogenic Mimicry (VM) was put forward, that people realized that in tumor tissue, the blood vessels made up of endothelial cells were found in the tumor tissue. There are other forms of blood supply. Vascular mimicry is a function of a tumor cell that mimics the formation of vascular endothelial cells in a specific condition, a function similar to the normal blood vessel, which contains red cells, and can connect with the conventional endothelial blood vessels to form a reticular circulation of blood, thus providing the necessary camp for the tumor. Support. It has been proved that vascular mimicry exists in various malignant tumors, including glioma, and the positive rate is closely related to the tumor grade and the poor prognosis of the patients. Therefore, the targeted vascular mimicry therapy strategy is likely to be a very promising method of anti swelling tumor vascular treatment, therefore, in the patients with glioma. While traditional antivascular therapy is carried out, the combined application of drugs to inhibit the formation of tumor vasculature may have a better therapeutic effect. More and more studies have shown that the tumor microenvironment plays an indispensable role in the formation of vascular mimicry, but the specific molecular mechanism has not yet been clearly defined and the tumor microenvironment will be deeply studied. Promote people to understand the mechanism related to vascular mimicry. Immune cell infiltration is a significant feature of various tumor microenvironments. It plays a key role in tumor invasion, angiogenesis, metastasis and other malignant biological behaviors, and then affects the progress of the tumor. It is found that the proportion of macrophages infiltrated in glioblastoma is up to 30%, and high expression of the M2 macrophage markers such as CD14, CD68, CD163 and CD204. Previous studies have found that the number and activity of local infiltrating macrophages in the tumor are closely related to the prognosis of the patients. In patients with recurrent glioblastoma, the number of infiltrating macrophages in the tumor increases significantly and is positively correlated with the tumor's blood vessel density. These results suggest that tumor infiltrating macrophages play an unneglecting role in the formation of tumor blood supply during the progression of the tumor. Therefore, the tumor infiltrates macrophages. Angiogenesis may be an effective way to overcome the antiangiogenic resistance of gliomas. Another important feature of the tumor microenvironment is the existence of a large number of unique immune mediators, including cytokines and chemokines. More and more data have shown that these immune mediators can regulate the work of immune cells and tumor cells. -6 (Interleukin-6, IL-6) is a kind of multifunctional immune medium, which is widely used in the inflammatory microenvironment of various malignant solid tumors. By participating in immunoregulation, it promotes tumor cell proliferation, migration, survival and drug resistance and causes cancer patients. In addition, the results show that IL-6 is inextricably related to the poor effect of tumor antiangiogenic therapy. In gliomas, IL-6 can promote tumor invasion and angiogenesis by enhancing the migration of vascular endothelial cells, blocking IL-6 and combining bavavic can more effectively impede the invasion and birth of glioma. These results suggest that the overexpression of IL-6 in the tumor microenvironment will significantly affect the angiogenesis of tumor, but it is still not clear whether IL-6 plays an important role in the formation of glioma angiogenesis. On the basis of the existing research, we should explore the relationship between vascular mimicry and tumor preliminarily by using immunohistochemical method. The expression of macrophages in human glioma tissue, clinical significance and the correlation between the two. Through in vitro experiments, it is analyzed whether THP-1 derived M2 macrophages and peripheral blood mononuclear derived M2 macrophages will affect the formation of glioma vascular mimicry, and explore its related mechanisms. Expression in glioma to detect the expression of vascular mimicry and CD163+ macrophage in glioma tissue, and to analyze the relationship between the two, and the relationship with the prognosis and the clinicopathological factors of the patients. Methods 1. the study was reviewed in the Qilu Hospital of Shandong University. The tumor tissue samples were obtained in 87 cases. Immunohistochemical method was used to detect the expression of vascular mimicry and CD163+ macrophage in glioma, and the correlation between the two.2. was analyzed and the basic information of the patients was collected, including basic clinical data such as sex and age, analysis of vascular mimicry and the presence of CD163+ macrophages and glioma patients. The relationship between pathological factors and prognosis. Results 1. CD31/PAS and CD163 staining showed that the level of vascular mimicry (CD31-/PAS+) and CD163 expression in high grade glioma (WHO grade III - IV) were significantly higher than those of lower grade (WHO grade I - II) (P0.01, P0.01) by immunohistochemical staining. The results showed that the level of vascular mimicry and the expression density of CD163 were positively correlated with the glioma tissue (r2=0.416, P0.001).2. analysis of the relationship between the vascular mimicry and the expression of CD163 in glioma tissue and the different pathological factors. The two were related to the level of the tumor (P0.001, P0.001), and the vascular mimicry was also related to the size of the tumor (P=0.027). .3.Kaplan-Meier analysis of sex, age, tumor location, KPS score, and tumor growth patterns found that the two had significant survival advantages compared to those with a higher level of CD163 expression in glioma patients with lower CD163 expression (p=0.006); the survival time of the patients with a low level of vascular mimicry was in the patients' survival time. Further analysis showed that the median survival time of patients with high expression of CD163 and high level glioma of vascular mimicry was significantly shorter than those of other groups of glioma (P0.001). Conclusion 1. the level of vascular mimicry and the number of macrophage infiltration in CD163+ are related to the grade of glioma, two The expression of.2.CD163+ macrophage infiltration and vascular mimicry in glioma tissues is associated with poor prognosis of patients with glioma, the level of.3. vascular mimicry and the number of CD163+ macrophages and the clinicopathological features of glioma patients, such as sex, age, tumor location, KPS score, and tumor growth patterns. Guan. Second part of THP-1 derived M2 macrophages regulating glioma vascular mimicry and mechanism study to explore whether THP-1 derived M2 macrophages affect the formation of glioma vascular mimicry and its possible mechanism method. 1. qRT-PCR method was used to detect the phenotype of THP-1 derived macrophages for the detection of THP-1 derived M2 macrophage fines Effects of cell on angiogenesis in glioma cells.3. using qRT-PCR, Western blotting and other methods to detect the effect of THP-1 derived M2 macrophages on the transcriptional and protein levels of vascular mimicry related markers in glioma cells.4. uses suspension chips and ELISA techniques to detect the IL-6 secretion of THP-1 derived M2 macrophages to glioma cells. The influence of THP-1 derived M2 macrophages on the expression time sequence of IL-6 and vascular mimicry in glioma cells by using qRT-PCR test technique,.6. was used as a tube test, Western blotting and other methods were used to detect the formation of.7. in the angiogenesis of glioma,.7., qRT-PCR, ELISA, and.5. The effect of THP-1 derived M2 macrophages on the related signal mechanism of glioma cells. Results 1.THP-1 derived M2 macrophages expressed high level M2 markers (CD163, CD206, IL-10, IL-1RA and CCL17) and low level M1 markers. The supernatant of the cell obviously promotes the formation of vascular mimicry in glioma cells (U251, P0.001; A172, P0.01) and vascular mimicry (MMP-9, MMP-14, LAMC2) expression of.3.THP-1 derived M2 macrophage supernatant to promote the secretion of IL-6 in glioma cells, which is characterized by time and concentration dependence, and IL-6 expression (approximately 6h) is earlier than that of vascular mimicry. The increased time (about 12h).4.IL-6 neutralizing antibody (1 mu g/ml) can block the expression of vascular mimicry induced by THP-1 derived M2 macrophages (U251, P0.01; A172, P0.01) and the up-regulated expression of related indexes. In addition, recombinant human IL-6 (rhIL-6100ng/ml) can promote the formation of vascular mimicry in glioma cells (U251, The expression of.01, A172, P0.05) and related indexes,.5.THP-1 derived M2 macrophage supernatant, the phosphorylation level.PKC inhibitor (Bisindolylmaleimide I) of glioma cell PKC signaling pathway can inhibit the transcription of glioma cells induced by THP-1 derived M2 macrophages. The expression of vascular mimicry in glioma cells (U251, P0.05; A172, P0.05) and the expression of related indexes. Conclusion 1.THP-1 derived M2 macrophages promote the formation of vascular mimicry of glioma cells in vitro, and the formation of.2.THP-1 derived M2 macrophages promotes the production of IL-6 by activating the glioma microcell PKC signaling pathway and then promotes the angiogenesis. Formation. Third partial monocyte derived M2 macrophages regulate glioma vascular mimicry and mechanism study to explore whether human peripheral blood mononuclear cells derived M2 macrophages affect the formation and mechanism of glioma vascular mimicry and mechanism method 1. separation of CD14+ mononuclear cells from human peripheral blood by magnetic bead separation, and the use of magnetic beads to separate CD14+ mononuclear cells from human peripheral blood .2. and ELISA techniques were used to detect the phenotypic.3. of human peripheral blood monocyte derived macrophages using qRT-PCR and ELISA techniques. The effects of M2 macrophages derived from human peripheral blood mononuclear cells on the secretion of IL-6 in human glioma cells were analyzed by qRT-PCR and ELISA methods. Effect of nuclear cell derived M2 macrophages on angiogenesis in glioma cells.5. use tube test to detect the effect of IL-6 on angiogenesis in glioma cells. Results 1. human peripheral blood monocyte derived M2 macrophages expressed high transcriptional level M2 markers (CD163, CD206, CCL18 and CCL17) and M of low transcriptional level. Type 1 markers (TNF-, IL-1 beta, IL-6, and IL-12). High level M2 markers secreted by monocyte derived M2 macrophages (CCL22, CCL18, CCL17 and IL-10) and low level M1 type markers 2, P0.001) and secreting (U251, P0.001; A172, P0.001) level.3. added IL-6 neutralization antibody (1 mu) to co culture system to significantly reduce the number of tubular structure of glioma cells (U251, P0.01; A172, P0.01). Conclusion: 1. peripheral blood monocyte derived M2 macrophages increase the expression of IL-6 in human glioma cells.2. human peripheral blood.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.41

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