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  本文選題：綠色裸小鼠　切入點(diǎn)：綠色熒光蛋白　出處：《蘇州大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：第一部分“綠色裸小鼠”腦組織中綠色熒光蛋白表達(dá)分析【目的】分析綠色熒光裸小鼠腦組織中綠色熒光蛋白(EGFP)的表達(dá)情況,為綠色裸小鼠的腦內(nèi)移植應(yīng)用提供依據(jù)。【方法】采用半定量RT-PCR分析綠色裸小鼠不同臟器組織/細(xì)胞和腦不同部位組織中EGFP m RNA表達(dá)水平;通過熒光顯微鏡觀察全身主要器官組織和全腦不同層面組織中綠色熒光強(qiáng)度;用熒光定量PCR和熒光分光光度計(jì)定量分析、免疫組化定性觀察鼠腦不同部位組織中EGFP的表達(dá);在綠色裸小鼠顱內(nèi)接種表達(dá)紅色熒光蛋白(RFP)的人膠質(zhì)瘤干祖細(xì)胞SU3-RFP,動物活體成像及熒光顯微鏡觀察小鼠活體腦和腦組織中腫瘤形成情況�！窘Y(jié)果】RT-PCR結(jié)果顯示,綠色裸小鼠各臟器組織和腦組織不同部位都不同程度表達(dá)EGFP;熒光顯微鏡下觀察到腦組織不同層面都能顯示綠色熒光,且呈現(xiàn)部位差異;q PCR和熒光分光光度計(jì)定量分析表明,與大腦皮質(zhì)相比,小腦皮層、海馬和嗅球等組織EGFP表達(dá)較高,差異具有顯著性(p0.05);免疫組化結(jié)果顯示,小腦皮層、海馬等部位存在強(qiáng)陽性表達(dá)EGFP的細(xì)胞;腫瘤細(xì)胞接種至綠色裸小鼠顱內(nèi)后,小動物活體成像儀和熒光顯微鏡下可見綠色腦組織背景下紅色熒光的腫瘤形成,表明成功建立了腦內(nèi)紅綠雙色熒光示蹤移植瘤模型。【結(jié)論】綠色熒光裸小鼠腦組織中不同程度表達(dá)EGFP,而非陰性表達(dá),其中小腦皮層、海馬和嗅球等部位表達(dá)較高。同其它高表達(dá)EGFP的臟器一樣,腦也適合做人癌移植熒光示蹤實(shí)驗(yàn)研究。第二部分雙色熒光示蹤移植瘤模型中骨髓來源宿主細(xì)胞惡性轉(zhuǎn)化初步研究【目的】利用紅綠雙色熒光示蹤移植瘤模型,探討腫瘤微環(huán)境中來源于宿主骨髓的間質(zhì)細(xì)胞的惡性轉(zhuǎn)化問題�！痉椒ā吭诰G色熒光示蹤骨髓重建模型基礎(chǔ)上,顱內(nèi)接種表達(dá)RFP膠質(zhì)瘤干祖細(xì)胞SU-RFP,建立紅綠雙色熒光示蹤移植瘤模型。分離移植瘤組織進(jìn)行體外培養(yǎng),對可連續(xù)傳代的發(fā)綠色熒光細(xì)胞進(jìn)行單克隆培養(yǎng)(命名為ih BTCBM),并對細(xì)胞生物學(xué)特征進(jìn)行分析:CCK-8法繪制細(xì)胞增殖曲線,常規(guī)方法進(jìn)行染色體核型分析;將細(xì)胞接種至裸小鼠皮下觀察細(xì)胞致瘤性;采用免疫熒光方法檢測細(xì)胞中骨髓間充質(zhì)干細(xì)胞標(biāo)準(zhǔn)物Sca-1、CD29、CD44表達(dá)情況。【結(jié)果】ih BTCBM細(xì)胞體外培養(yǎng)呈現(xiàn)出惡性細(xì)胞表型,細(xì)胞增殖旺盛,染色體為異倍體,皮下接種裸小鼠致瘤率為100%(6/6);免疫熒光檢測結(jié)果顯示細(xì)胞表達(dá)骨髓間充質(zhì)干細(xì)胞標(biāo)準(zhǔn)物Sca-1、CD29和CD44�！窘Y(jié)論】在EGFP熒光示蹤下,從雙色熒光示蹤移植瘤組織中分離出宿主骨髓來源、可連續(xù)傳代的細(xì)胞,該細(xì)胞可能是骨髓間充質(zhì)干細(xì)胞的惡性轉(zhuǎn)化的細(xì)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).
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.6

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 Himanshu Priyadarshi;Absar Alam;Gireesh-Babu P;Rekha Das;Pankaj Kishore;Shivendra Kumar;Aparna Chaudhari;;A GFP-based bacterial biosensor with chromosomally integrated sensing cassette for quantitative detection of Hg(Ⅱ) in environment[J];Journal of Environmental Sciences;2012年05期

2 吳沛橋;巴曉革;胡海;趙靜;;綠色熒光蛋白GFP的研究進(jìn)展及應(yīng)用[J];生物醫(yī)學(xué)工程研究;2009年01期

3 銀廣悅;陳素萍;丁俊麗;張繼領(lǐng);劉繼勇;張龍;張明;;小鼠骨髓間充質(zhì)干細(xì)胞的體外分離培養(yǎng)和鑒定方法學(xué)探討[J];中國實(shí)驗(yàn)診斷學(xué);2013年04期

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