【摘要】：目的：建立紅、綠雙色熒光示蹤的膠質(zhì)瘤原位移植模型，并分析其實(shí)用價(jià)值。 方法：將發(fā)紅色熒光的C6膠質(zhì)瘤細(xì)胞接種于綠色熒光裸小鼠腦內(nèi)，動(dòng)態(tài)活體熒光成像。實(shí)驗(yàn)結(jié)束時(shí)，取全腦做連續(xù)冰凍切片，在普通光鏡、熒光顯微鏡和激光共聚焦顯微鏡下觀察移植瘤組織結(jié)構(gòu)。 結(jié)果：活體熒光顯像分析顯示，接種腫瘤細(xì)胞的相應(yīng)部位均可見(jiàn)到發(fā)紅色熒光的腫瘤團(tuán)塊影存在，并且其大小隨著腫瘤移植時(shí)間的延長(zhǎng)而增大。在移植瘤組織冰凍切片中，可清晰顯示腫瘤組織中存在紅色、綠色和黃色等3種顏色的熒光細(xì)胞，并可在不同部位界定3種熒光細(xì)胞之間的位置關(guān)系；腫瘤細(xì)胞遷徙、定植及其與宿主細(xì)胞的融合等清晰可見(jiàn)；根據(jù)熒光顏色可以鑒別組成腫瘤血管的起源細(xì)胞是宿主細(xì)胞(綠色)、腫瘤細(xì)胞(紅色)或者是腫瘤細(xì)胞與宿主細(xì)胞融合(黃色)。此外，腫瘤周邊微環(huán)境中宿主固有的星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞被激活和去分化成Nestin陽(yáng)性細(xì)胞。 結(jié)論：與傳統(tǒng)模型比較，雙色熒光示蹤的膠質(zhì)瘤原位移植模型在腫瘤活體成像及研究腫瘤細(xì)胞侵襲、轉(zhuǎn)移、腫瘤血管生成和腫瘤周邊微環(huán)境中宿主固有細(xì)胞被激活等方面，具有更高的實(shí)用價(jià)值。 目的：課題組前期研究顯示膠質(zhì)瘤腫瘤間質(zhì)中源于宿主的細(xì)胞可被膠質(zhì)瘤干祖細(xì)胞誘導(dǎo)惡變，但這些惡性轉(zhuǎn)化細(xì)胞對(duì)射線是否與膠質(zhì)瘤干祖細(xì)胞一樣高度抵抗尚不明確，，本文旨在初步探索這些惡變細(xì)胞對(duì)射線的敏感性及可能的分子機(jī)制。 方法：連續(xù)多次輻射誘導(dǎo)，篩選并建立比膠質(zhì)瘤干祖細(xì)胞SU3更加耐輻射的SU3-5R細(xì)胞；分別將SU3、SU3-5R及SU3誘導(dǎo)惡變的宿主少突膠質(zhì)細(xì)胞ihBTC2輻射，計(jì)算克隆形成率，繪制各細(xì)胞株放射劑量-存活曲線；熒光定量PCR檢測(cè)各細(xì)胞株輻射后Notch1mRNA及Hes1mRNA的表達(dá)水平；Western blot檢測(cè)ihBTC2細(xì)胞Notch信號(hào)通路下游輻射相關(guān)蛋白Bcl-2及pAkt表達(dá)水平。 結(jié)果：膠質(zhì)瘤間質(zhì)宿主惡變細(xì)胞ihBTC2比耐輻射細(xì)胞SU3-5R更加輻射抵抗，表現(xiàn)為細(xì)胞增殖快，SF2值較高，輻射誘導(dǎo)性細(xì)胞凋亡壞死較低，輻射后Notch1、pAkt、Bcl-2表達(dá)水平顯著增高，而采用γ-分泌酶抑制劑（GSIs）阻斷Notch信號(hào)通路可提高其對(duì)射線的敏感性。 結(jié)論：在膠質(zhì)瘤微環(huán)境中，腫瘤間質(zhì)惡變的宿主少突膠質(zhì)細(xì)胞ihBTC2對(duì)射線更加抵抗，可能與Notch信號(hào)通路激活密切相關(guān)，這對(duì)進(jìn)一步研究腫瘤間質(zhì)細(xì)胞與腫瘤放療敏感性之間的關(guān)系有重要價(jià)值，為進(jìn)一步探索膠質(zhì)瘤耐輻射的新機(jī)制提供參考依據(jù)。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.41

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