發(fā)布時(shí)間：2018-03-09 11:51

  本文選題：標(biāo)記滯留細(xì)胞　切入點(diǎn)：轉(zhuǎn)基因小鼠　出處：《華中科技大學(xué)》2011年博士論文　論文類型：學(xué)位論文

【摘要】：目的：建立小鼠尿路上皮“標(biāo)記滯留細(xì)胞”的動(dòng)物模型,尋找小鼠尿路上皮干細(xì)胞的體內(nèi)定位。方法：將轉(zhuǎn)基因小鼠Krt5-rtTa-VP16與TRE-H2B-GFP雜交,獲得雙轉(zhuǎn)基因小鼠。Krt5,GFP等基因的表達(dá)用RT-PCR技術(shù)鑒定。待此小鼠長(zhǎng)到4周后進(jìn)行脈沖追蹤(pulse-chase)實(shí)驗(yàn),喂予含強(qiáng)力霉素的食物,并注射環(huán)磷酰胺以刺激尿路上皮增殖,2周后獲得尿路上皮基底層細(xì)胞表達(dá)綠色熒光蛋白(GFP)的雙轉(zhuǎn)基因小鼠。Chase期后制作組織連續(xù)切片和組織整標(biāo)本,用表面熒光顯微鏡,立式顯微鏡,三維反卷積顯微鏡和雙光子共聚焦顯微鏡觀察GFP標(biāo)記滯留細(xì)胞在小鼠尿路系統(tǒng)中的分布。用Image J, Volocity等軟件對(duì)圖像及數(shù)據(jù)進(jìn)行重建和整合。結(jié)果：轉(zhuǎn)基因小鼠Krt5-rtTa-VP16與TRE-H2B-GFP雜交后獲得雙轉(zhuǎn)基因小鼠Krt5-rtTa/TRE-H2B-GFP。此小鼠進(jìn)食含強(qiáng)力霉素的食物后尿路上皮基底層表達(dá)綠色熒光蛋白。小鼠膀胱,輸尿管連續(xù)切片的結(jié)果顯示,含GFP的標(biāo)記滯留細(xì)胞分布于膀胱三角區(qū),膀胱頸,腎盂輸尿管連接處和輸尿管膀胱連接處。對(duì)小鼠膀胱和輸尿管整標(biāo)本采集的數(shù)據(jù)基本與連續(xù)切片的結(jié)果吻合。結(jié)論：此實(shí)驗(yàn)成功建立了鑒定小鼠尿路上皮干細(xì)胞的方法,觀察到小鼠尿路上皮干細(xì)胞分布于膀胱三角區(qū),膀胱頸,腎盂輸尿管連接處和輸尿管膀胱連接處,為上皮干細(xì)胞的進(jìn)一步研究打下基礎(chǔ),同時(shí)也為尿路腫瘤的研究提供了理論依據(jù)。
[Abstract]:Objective: to establish an animal model of "labeled retention cells" of mouse urinary tract epithelium, and to find out the location of mouse urinary tract epithelial stem cells in vivo. Methods: Krt5-rtTa-VP16 and TRE-H2B-GFP were hybridized in transgenic mice. The expression of .Krt5GFP and other genes was identified by RT-PCR technique. After the mice reached 4 weeks old, pulse-chasetest was carried out, and the mice were fed with Doxycycline (Doxycycline). After injection of cyclophosphamide to stimulate the proliferation of urinary tract epithelium, two weeks later, the double transgenic mice, which expressed green fluorescent protein (GFP) in basal layer cells of urinary tract epithelium, were prepared tissue serial sections and whole tissue samples after phase. Vertical microscope, Three-dimensional deconvolution microscope and two-photon confocal microscope were used to observe the distribution of GFP labeled retained cells in mouse urinary tract system. Images and data were reconstructed and integrated with Image J, Volocity and other software. Results: transgenic mice Krt5-rtTa-VP16 and. After TRE-H2B-GFP hybridization, two transgenic mice, Krt5-rtTa-TRE-H2B-GFP.The mouse urinary tract epithelium expressed green fluorescent protein after eating the food containing doxycycline. The results of ureteral serial sections showed that the labeled retained cells containing GFP were located in the bladder triangle, bladder neck, and bladder neck. The data collected from the whole sample of mouse bladder and ureter were basically consistent with the results of serial sections. Conclusion: the method of identifying mouse urothelial stem cells was successfully established in this experiment. It was observed that the epithelial stem cells in the urinary tract of mice were located in the bladder triangle, bladder neck, ureteropelvic junction and ureterocervical junction, which laid the foundation for the further study of the epithelial stem cells. It also provides a theoretical basis for the study of urinary tract tumors.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

【共引文獻(xiàn)】

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相關(guān)博士學(xué)位論文 前2條

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相關(guān)碩士學(xué)位論文 前6條

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本文編號(hào)：1588407