双色荧光示踪胶质瘤原位移植模型的建立及其恶变的肿瘤间质细胞耐辐射特性的研究

发布时间：2019-07-05 11:30

【摘要】：目的：建立红、绿双色荧光示踪的胶质瘤原位移植模型，并分析其实用价值。方法：将发红色荧光的C6胶质瘤细胞接种于绿色荧光裸小鼠脑内，动态活体荧光成像。实验结束时，取全脑做连续冰冻切片，在普通光镜、荧光显微镜和激光共聚焦显微镜下观察移植瘤组织结构。结果：活体荧光显像分析显示，接种肿瘤细胞的相应部位均可见到发红色荧光的肿瘤团块影存在，并且其大小随着肿瘤移植时间的延长而增大。在移植瘤组织冰冻切片中，可清晰显示肿瘤组织中存在红色、绿色和黄色等3种颜色的荧光细胞，并可在不同部位界定3种荧光细胞之间的位置关系；肿瘤细胞迁徙、定植及其与宿主细胞的融合等清晰可见；根据荧光颜色可以鉴别组成肿瘤血管的起源细胞是宿主细胞(绿色)、肿瘤细胞(红色)或者是肿瘤细胞与宿主细胞融合(黄色)。此外，肿瘤周边微环境中宿主固有的星形胶质细胞和少突胶质细胞被激活和去分化成Nestin阳性细胞。结论：与传统模型比较，双色荧光示踪的胶质瘤原位移植模型在肿瘤活体成像及研究肿瘤细胞侵袭、转移、肿瘤血管生成和肿瘤周边微环境中宿主固有细胞被激活等方面，具有更高的实用价值。目的：课题组前期研究显示胶质瘤肿瘤间质中源于宿主的细胞可被胶质瘤干祖细胞诱导恶变，但这些恶性转化细胞对射线是否与胶质瘤干祖细胞一样高度抵抗尚不明确，，本文旨在初步探索这些恶变细胞对射线的敏感性及可能的分子机制。方法：连续多次辐射诱导，筛选并建立比胶质瘤干祖细胞SU3更加耐辐射的SU3-5R细胞；分别将SU3、SU3-5R及SU3诱导恶变的宿主少突胶质细胞ihBTC2辐射，计算克隆形成率，绘制各细胞株放射剂量-存活曲线；荧光定量PCR检测各细胞株辐射后Notch1mRNA及Hes1mRNA的表达水平；Western blot检测ihBTC2细胞Notch信号通路下游辐射相关蛋白Bcl-2及pAkt表达水平。结果：胶质瘤间质宿主恶变细胞ihBTC2比耐辐射细胞SU3-5R更加辐射抵抗，表现为细胞增殖快，SF2值较高，辐射诱导性细胞凋亡坏死较低，辐射后Notch1、pAkt、Bcl-2表达水平显著增高，而采用γ-分泌酶抑制剂（GSIs）阻断Notch信号通路可提高其对射线的敏感性。结论：在胶质瘤微环境中，肿瘤间质恶变的宿主少突胶质细胞ihBTC2对射线更加抵抗，可能与Notch信号通路激活密切相关，这对进一步研究肿瘤间质细胞与肿瘤放疗敏感性之间的关系有重要价值，为进一步探索胶质瘤耐辐射的新机制提供参考依据。
[Abstract]:Objective: to establish an orthotopic transplantation model of glioma with red and green fluorescence tracer and analyze its practical value. Methods: C 6 glioma cells with red fluorescence were inoculated into the brain of green fluorescent nude mice, and dynamic fluorescence imaging was performed in vivo. At the end of the experiment, the whole brain was taken for continuous frozen sections, and the tissue structure of the transplanted tumor was observed under ordinary light microscope, fluorescence microscope and laser confocal microscope. Results: in vivo fluorescence imaging analysis showed that red fluorescent tumor masses could be seen in the corresponding parts of inoculated tumor cells, and their size increased with the prolongation of tumor transplantation time. In the frozen section of the tumor tissue, the fluorescent cells of three colors, such as red, green and yellow, could be clearly displayed in the tumor tissue, and the position relationship among the three fluorescent cells could be defined in different parts, and the migration, colonization and fusion of the tumor cells with the host cells could be clearly seen. According to the fluorescence color, the origin cells that make up the tumor blood vessels can be identified as host cells (green), tumor cells (red) or fusion of tumor cells and host cells (yellow). In addition, astrocytes and oligodendrocytes were activated and dedifferentiated into Nestin positive cells in the surrounding microenvironment of the tumor. Conclusion: compared with the traditional model, the two-color fluorescence tracer glioma orthotopic transplantation model has higher practical value in the imaging of tumor cells in vivo and in the study of tumor cell invasion, metastasis, tumor angiogenesis and activation of host intrinsic cells in the surrounding microenvironment. Aim: previous studies have shown that the cells from the host in the stroma of glioma tumors can be induced by glioma stem progenitor cells, but it is not clear whether these malignant transformed cells are as resistant to radiation as glioma stem progenitor cells. The purpose of this study is to explore the sensitivity of these malignant cells to radiation and the possible molecular mechanism. Methods: SU3-5R cells, which were more resistant to radiation than glioma stem progenitor cells SU3, were irradiated with SU3,SU3-5R and SU3 induced malignant host oligodendrocytes, the clone formation rate was calculated, the radiation dose-survival curves of each cell line were drawn, and the expression levels of Notch1mRNA and Hes1mRNA were detected by fluorescence quantitative PCR. The expression of radiation-related proteins Bcl-2 and pAkt downstream of Notch signaling pathway in ihBTC2 cells was detected by Western blot. Results: ihBTC2 of malignant host cells of glioma was more resistant to radiation than SU3-5R of radiation-resistant cells, which showed that cell proliferation was faster, SF _ 2 value was higher, apoptosis and necrosis of radiation-induced cells were lower, and the expression level of Notch1,pAkt,Bcl-2 was significantly increased after radiation. (GSIs), a gamma-secretase inhibitor, could increase the sensitivity of Notch signal pathway to radiation. Conclusion: in glioma microenvironment, oligodendrocytes ihBTC2, which is more resistant to radiation in glioma microenvironment, may be closely related to the activation of Notch signaling pathway, which is of great value to the further study of the relationship between tumor Leydig cells and tumor radiotherapy sensitivity, and provides a reference for further exploring the new mechanism of radiation tolerance in gliomas.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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