小鼠小肠干细胞谱系追踪方法的建立及其在放射损伤中的应用

发布时间：2018-08-11 17:22

【摘要】：目的利用Lgr5-EGFP-Cre ERT2/ROSA26-stop-EYFP小鼠建立谱系追踪方法,用于观察Lgr5+小肠干细胞在小肠组织更新中的作用,并研究辐射对小肠干细胞组织重建的影响。方法通过基因型分析鉴定Lgr5-EGFP-Cre ERT2/ROSA26-stop-EYFP小鼠。他莫西芬诱导小鼠体内的荧光蛋白表达,激光共聚焦显微镜观察诱导后小肠干细胞的分化。采用60Coγ射线(15Gy)照射Lgr5-EGFP-Cre ERT2/ROSA26-stop-EYFP小鼠,观察辐射对小肠干细胞组织重建的影响。结果提取鼠尾DNA通过PCR方法鉴定Lgr5基因(174bp),获得双阳性小鼠。他莫西芬(100mg/kg)单次诱导后,激光共聚焦观察到Lgr5+小肠干细胞在诱导后5d开始分裂,7d已到达绒毛顶端,14d少量绒毛全部带有荧光,45d更多的小肠绒毛中存在荧光细胞,而且更加稠密。但在15Gy照射后小鼠小肠绒毛脱落严重,隐窝处没有荧光出现。结论成功建立了小肠干细胞谱系追踪模型,并初步观察了辐射对小肠干细胞损伤的影响。
[Abstract]:Objective to establish a lineage tracing method in Lgr5-EGFP-Cre ERT2/ROSA26-stop-EYFP mice to observe the role of Lgr5 small intestinal stem cells in small intestinal tissue regeneration and to study the effects of radiation on small intestinal stem cell tissue reconstruction. Methods Lgr5-EGFP-Cre ERT2/ROSA26-stop-EYFP mice were identified by genotype analysis. The expression of fluorescent protein was induced by tamoxifen and the differentiation of small intestinal stem cells was observed by laser confocal microscopy. Lgr5-EGFP-Cre ERT2/ROSA26-stop-EYFP mice were irradiated with 60Co 纬-ray (15Gy) to observe the effect of radiation on the tissue reconstruction of small intestinal stem cells. Results DNA was extracted from mouse tail to identify Lgr5 gene (174bp) by PCR method, and double positive mice were obtained. After a single induction by tamoxifen (100mg/kg), Lgr5 small intestinal stem cells were observed to divide at 5 days after induction and reached the top of the villi for 14 days. A small number of villi were found to have fluorescent cells in the villi of the small intestine with fluorescence for 45 days and more dense. However, after 15Gy irradiation, the villi of the small intestine fell off seriously and there was no fluorescence in the crypt. Conclusion the small intestinal stem cell lineage tracing model was successfully established and the effects of radiation on the injury of small intestinal stem cells were preliminarily observed.
【作者单位】：广西医科大学研究生院;军事医学科学院放射与辐射医学研究所;
【基金】：国家重大新药创制专项课题(2013ZX09J13102-02C) 国家自然科学基金面上项目(81272997)~~
【分类号】：R818

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