尿路上皮干细胞的鉴定

发布时间：2018-03-09 11:51

本文选题：标记滞留细胞　切入点：转基因小鼠　出处：《华中科技大学》2011年博士论文　论文类型：学位论文

【摘要】：目的：建立小鼠尿路上皮“标记滞留细胞”的动物模型,寻找小鼠尿路上皮干细胞的体内定位。方法：将转基因小鼠Krt5-rtTa-VP16与TRE-H2B-GFP杂交,获得双转基因小鼠。Krt5,GFP等基因的表达用RT-PCR技术鉴定。待此小鼠长到4周后进行脉冲追踪(pulse-chase)实验,喂予含强力霉素的食物,并注射环磷酰胺以刺激尿路上皮增殖,2周后获得尿路上皮基底层细胞表达绿色荧光蛋白(GFP)的双转基因小鼠。Chase期后制作组织连续切片和组织整标本,用表面荧光显微镜,立式显微镜,三维反卷积显微镜和双光子共聚焦显微镜观察GFP标记滞留细胞在小鼠尿路系统中的分布。用Image J, Volocity等软件对图像及数据进行重建和整合。结果：转基因小鼠Krt5-rtTa-VP16与TRE-H2B-GFP杂交后获得双转基因小鼠Krt5-rtTa/TRE-H2B-GFP。此小鼠进食含强力霉素的食物后尿路上皮基底层表达绿色荧光蛋白。小鼠膀胱,输尿管连续切片的结果显示,含GFP的标记滞留细胞分布于膀胱三角区,膀胱颈,肾盂输尿管连接处和输尿管膀胱连接处。对小鼠膀胱和输尿管整标本采集的数据基本与连续切片的结果吻合。结论：此实验成功建立了鉴定小鼠尿路上皮干细胞的方法,观察到小鼠尿路上皮干细胞分布于膀胱三角区,膀胱颈,肾盂输尿管连接处和输尿管膀胱连接处,为上皮干细胞的进一步研究打下基础,同时也为尿路肿瘤的研究提供了理论依据。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329

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