巨噬细胞诱导胶质瘤干细胞促血管生成作用的初步研究

发布时间：2018-05-10 14:40

本文选题：血管生成 + 胶质瘤干细胞　；参考：《蚌埠医学院》2014年硕士论文

【摘要】：恶性胶质细胞瘤是中枢神经系统最常见的原发性肿瘤，而且是人体血管化程度最高的肿瘤之一。胶质瘤的血管生成是其生长和侵袭的基础，从而影响患者的放、化疗效果，导致其死亡率居高不下。肿瘤干细胞(cancer stem cells,CSCs)在肿瘤发生和发展中的作用越来越受到人们的重视。我科在前期工作中已对胶质瘤干细胞(glioma stem cells, GSCs)有了较为系统和深入的研究，发现了CD133可以作为GSCs的特异标志物，并阐明CD133阳性的GSCs可以自我更新和多向分化，且具有很强的侵袭能力。已有文献报道肿瘤微环境可以影响肿瘤的发生和发展。例如在肿瘤的早期，其组织中就有大量炎症细胞的浸润，其中巨噬细胞占有较高的比例，且参与肿瘤发生及演进的诸多方面，但是关于巨噬细胞对胶质瘤干细胞促血管生成的影响有待深入研究。目前，在胶质瘤血管生成的研究中，，研究者大多采用体外培养方式进行，因此无法在完整的活体中直观地观察巨噬细胞对胶质瘤干细胞促血管生成的作用。斑马鱼作为一种模式生物在肿瘤研究中已被广泛应用。研究报道，斑马鱼体内的巨噬细胞是最早出现的免疫细胞，再加之，fli:GFP转基因斑马鱼的建立，使其全部血管被标记了绿色荧光蛋白，从而使人们能够直观地观察到活体斑马鱼的血管生成情况。因此，应用斑马鱼模型探讨巨噬细胞在肿瘤发生发展的早期是如何影响胶质瘤干细胞促血管生成的作用具有重要意义。本研究首先将人胶质瘤原代细胞091214转染红色荧光蛋白（RFP），再以CD133作为标志物，将转有红色荧光蛋白的GSCs分选出来；将分选出的CD133+和CD133-的胶质瘤细胞移植入fli:GFP转基因斑马鱼胚胎的卵黄囊区域，我们可以发现GSCs在斑马鱼体内具有明显的促血管生成的作用；然后利用中性红染色的方法对活体斑马鱼的巨噬细胞进行标记，以研究巨噬细胞对GSCs促血管生成的影响；最后，我们又做了斑马鱼巨噬细胞特异性基因(L-plastin)的整体原位杂交实验，以期在mRNA水平对巨噬细胞进行定位，从而更准确地研究巨噬细胞在GSCs促血管生成中的作用。综上所述，本研究建立了胶质瘤干细胞在斑马鱼诱导血管生成的体内模型，发现巨噬细胞在血管生成部位的分布与胶质瘤干细胞的促血管生成作用具有密切关系，表明巨噬细胞的浸润促进了胶质瘤干细胞诱导的血管生成。本实验为基于巨噬细胞参与的促肿瘤血管生成的靶向治疗提供了实验依据。
[Abstract]:Malignant glioma is the most common primary tumor in the central nervous system and one of the most vascularized tumors. Angiogenesis is the basis of glioma growth and invasion, which affects the effect of radiotherapy and chemotherapy, resulting in high mortality. More and more attention has been paid to the role of cancer stem cell stem cells in tumorigenesis and development. In our previous work, we have made a systematic and in-depth study on glioma stem cells, GSCs), and found that CD133 can be used as a specific marker of GSCs, and that CD133 positive GSCs can be self-renewing and multi-differentiated. And has the very strong invasiveness. It has been reported that tumor microenvironment can affect the occurrence and development of tumor. For example, in the early stage of tumor, there are a large number of inflammatory cells infiltrated in its tissues, in which macrophages occupy a high proportion, and are involved in many aspects of tumorigenesis and progression. However, the effects of macrophages on angiogenesis of glioma stem cells need further study. At present, in the study of glioma angiogenesis, most of the researchers use in vitro culture, so it is impossible to directly observe the role of macrophages in promoting angiogenesis of glioma stem cells in vivo. Zebrafish as a model organism has been widely used in tumor research. The study reported that the macrophages in zebrafish were the first immunocytes, and the establishment of flifish: GFP transgenic zebrafish made all its blood vessels labeled with green fluorescent protein (GFP). Thus, the angiogenesis of living zebrafish can be observed intuitively. Therefore, it is important to explore how macrophages affect the angiogenesis of glioma stem cells in the early stage of tumor development using zebrafish model. In this study, the primary human glioma cell line 091214 was transfected with the red fluorescent protein (CD133), and then the GSCs transformed with the red fluorescent protein was selected as a marker. The selected CD133 and CD133- glioma cells were transplanted into the yolk sac region of fli:GFP transgenic zebrafish embryos. We can find that GSCs plays a significant role in promoting angiogenesis in zebrafish. Then the macrophages of zebrafish in vivo were labeled by neutral red staining to study the effect of macrophages on angiogenesis induced by GSCs. Finally, in situ hybridization of the macrophage specific gene (L-plastin) of zebrafish was carried out. The aim of this study was to locate macrophages at mRNA level and to study the role of macrophages in angiogenesis induced by GSCs. In conclusion, a model of glioma stem cells induced angiogenesis in zebrafish was established in this study. It was found that the distribution of macrophages in the angiogenesis site was closely related to the angiogenesis of glioma stem cells. This suggests that macrophage infiltration promotes angiogenesis induced by glioma stem cells. This study provides experimental evidence for the targeting therapy of tumor angiogenesis based on macrophage involvement.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

【共引文献】