绿色裸小鼠中绿色荧光蛋白荧光示踪作用研究

发布时间：2018-03-19 15:28

本文选题：绿色裸小鼠　切入点：绿色荧光蛋白　出处：《苏州大学》2015年硕士论文　论文类型：学位论文

【摘要】：第一部分“绿色裸小鼠”脑组织中绿色荧光蛋白表达分析【目的】分析绿色荧光裸小鼠脑组织中绿色荧光蛋白(EGFP)的表达情况,为绿色裸小鼠的脑内移植应用提供依据。【方法】采用半定量RT-PCR分析绿色裸小鼠不同脏器组织/细胞和脑不同部位组织中EGFP m RNA表达水平;通过荧光显微镜观察全身主要器官组织和全脑不同层面组织中绿色荧光强度;用荧光定量PCR和荧光分光光度计定量分析、免疫组化定性观察鼠脑不同部位组织中EGFP的表达;在绿色裸小鼠颅内接种表达红色荧光蛋白(RFP)的人胶质瘤干祖细胞SU3-RFP,动物活体成像及荧光显微镜观察小鼠活体脑和脑组织中肿瘤形成情况。【结果】RT-PCR结果显示,绿色裸小鼠各脏器组织和脑组织不同部位都不同程度表达EGFP;荧光显微镜下观察到脑组织不同层面都能显示绿色荧光,且呈现部位差异;q PCR和荧光分光光度计定量分析表明,与大脑皮质相比,小脑皮层、海马和嗅球等组织EGFP表达较高,差异具有显著性(p0.05);免疫组化结果显示,小脑皮层、海马等部位存在强阳性表达EGFP的细胞;肿瘤细胞接种至绿色裸小鼠颅内后,小动物活体成像仪和荧光显微镜下可见绿色脑组织背景下红色荧光的肿瘤形成,表明成功建立了脑内红绿双色荧光示踪移植瘤模型。【结论】绿色荧光裸小鼠脑组织中不同程度表达EGFP,而非阴性表达,其中小脑皮层、海马和嗅球等部位表达较高。同其它高表达EGFP的脏器一样,脑也适合做人癌移植荧光示踪实验研究。第二部分双色荧光示踪移植瘤模型中骨髓来源宿主细胞恶性转化初步研究【目的】利用红绿双色荧光示踪移植瘤模型,探讨肿瘤微环境中来源于宿主骨髓的间质细胞的恶性转化问题。【方法】在绿色荧光示踪骨髓重建模型基础上,颅内接种表达RFP胶质瘤干祖细胞SU-RFP,建立红绿双色荧光示踪移植瘤模型。分离移植瘤组织进行体外培养,对可连续传代的发绿色荧光细胞进行单克隆培养(命名为ih BTCBM),并对细胞生物学特征进行分析:CCK-8法绘制细胞增殖曲线,常规方法进行染色体核型分析;将细胞接种至裸小鼠皮下观察细胞致瘤性;采用免疫荧光方法检测细胞中骨髓间充质干细胞标准物Sca-1、CD29、CD44表达情况。【结果】ih BTCBM细胞体外培养呈现出恶性细胞表型,细胞增殖旺盛,染色体为异倍体,皮下接种裸小鼠致瘤率为100%(6/6);免疫荧光检测结果显示细胞表达骨髓间充质干细胞标准物Sca-1、CD29和CD44。【结论】在EGFP荧光示踪下,从双色荧光示踪移植瘤组织中分离出宿主骨髓来源、可连续传代的细胞,该细胞可能是骨髓间充质干细胞的恶性转化的细胞。
[Abstract]:The first part is the analysis of the expression of green fluorescent protein (EGFP) in the brain tissue of "green nude mice" [objective] to analyze the expression of green fluorescent protein (EGFP) in the brain of green naked mice. [methods] Semi-quantitative RT-PCR was used to analyze the expression of EGFP m RNA in different organs / cells and brain tissues of green nude mice. The green fluorescence intensity was observed by fluorescence microscope in the main organs of the whole body and at different levels of the brain, and the expression of EGFP in different parts of the brain was detected qualitatively by immunohistochemistry and quantitative analysis by fluorescence quantitative PCR and fluorescence spectrophotometer. Human glioma stem progenitor cells SU3-RFP were inoculated into the brain of green nude mice. Animal imaging and fluorescence microscopy were used to observe the tumor formation in mouse brain and brain tissue. [results] RT-PCR results showed that, EGFP was expressed in different parts of organs and brain tissues of green nude mice to different degrees, green fluorescence was observed in different layers of brain tissue under fluorescence microscope, and quantitative analysis of site difference Q PCR and fluorescence spectrophotometer showed that EGFP could be detected in different layers of brain tissue. The expression of EGFP in cerebellar cortex, hippocampus and olfactory bulb was higher than that in cerebral cortex (P 0.05). After the tumor cells were inoculated into the brain of the green nude mice, the formation of red fluorescent tumors in the background of green brain tissue was observed under the living animal imager and fluorescence microscope. [conclusion] EGFP was expressed in the brain tissue of green green nude mice, but not negative, in which the cerebellar cortex, EGFP was expressed in the brain of the nude mice, and the tumor model was successfully established. [conclusion] EGFP was expressed in the brain tissue of the green fluorescent nude mice, but not the negative expression. The hippocampus and olfactory bulb are highly expressed. Like other organs with high expression of EGFP, The brain is also suitable for the study of human cancer transplantation with fluorescence tracer. The second part is the primary study of malignant transformation of bone marrow derived host cells in the model of transplanted tumor with two-color fluorescence tracer. [objective] to use red and green dual color fluorescent tracer to trace the tumor model. To study the malignant transformation of mesenchymal cells from host bone marrow in tumor microenvironment. [methods] based on the green fluorescent tracing bone marrow reconstruction model, RFP glioma stem progenitor cells (SU-RFP) were inoculated into the brain to establish a red and green bichromatic fluorescent tracer tumor model. The transplanted tumor tissues were isolated and cultured in vitro. The hair green fluorescent cells were cultured by monoclonal method (named ih BTCBM), and the cell proliferation curve was drawn by the method of cell biological analysis with the method of: CCK-8, and the karyotype of chromosome was analyzed by routine method. The cells were inoculated into nude mice subcutaneously to observe the tumorigenicity of the cells, and the expression of Sca-1 CD29 and CD44 in bone marrow mesenchymal stem cells was detected by immunofluorescence method. [results] the culture of Ih BTCBM cells in vitro showed malignant cell phenotype, and the proliferation of the cells was strong. The chromosome was aneuploidy, and the tumorigenic rate of nude mice was 100%. Immunofluorescence assay showed that the cells expressed Sca-1 CD29 and CD44. [conclusion] in the EGFP fluorescence tracer, Sca-1 CD29 and CD44 were expressed in the cells. [conclusion] under the EGFP fluorescence tracer, the cells expressed the bone marrow mesenchymal stem cell standard Sca-1 CD29 and CD44. The cells derived from the host bone marrow were isolated from the tumor tissue with double color fluorescence tracer. The cells could be the malignant transformation cells of the bone marrow mesenchymal stem cells (BMSCs).
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6

【参考文献】