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  

Establishment of lineage tracing method of mice small intestinal stem cell and application in radiation injury

作　　者：徐铁柱[1] 崔宇[2] 杨超[2] 王丽梅[2] 李中堂[2] 王俊可 赵致维 善亚君[2] 柳晓兰[2] 从玉文[2] 

机构地区：[1]广西医科大学研究生院,南宁530000 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《解放军医学杂志》2015年第5期349-353,共5页Medical Journal of Chinese People's Liberation Army

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)~~

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。Objective To establish a lineage tracing method with Lgr5-EGFP-Cre ERT2/ROSA26-stop-EYFP mouse for observing the role of Lgr5＋ intestinal stem cells in the renovation of small intestine tissue, and investigate the effects of radiation on the reconstruction of small intestinal stem cell tissue. Methods Lgr5-EGFP-Cre ERT2/ROSA26-stop-EYFP mice were identified by genotype analysis. Tamoxifen was used to induce the expression of fluorescent protein in mice. The differentiation of intestinal stem cells induced by Tamoxifen was observed by laser confocal microscopy. Lgr5-EGFP-Cre ERT2/ROSA26-stop-EYFP mice were irradiated with 60 Co γ-rays, and the effects of irradiation on the reconstruction of small intestinal stem cell tissue were examined. Results Double positive mice were obtained by identification of Lgr5 gene（174bp） in DNA extraction from the mice tail with PCR method. In mice treated with single injection of Tamoxifen（100mg/kg）, it was observed by laser confocal scanning microscopy that the Lgr5＋ intestinal stem cells started dividing at the 5th day after inducement, reached the top of the villi at the 7th day, fluorescence appeared in a few of whole intestinal villi at the 14 th day, and spread over more intestinal villi with dense fluorescence at the 45 th day. However, in mice exposed to 15 Gy irradiation, the intestinal villi fell off seriously without fluorescence in crypts. Conclusion The lineage tracing model of intestinal stem cell has been successfully established and then used to evaluate the irradiation injuries to intestinal stem cells.

关 键 词：小肠干细胞 谱系追踪 辐射损伤 

分 类 号：R818.04[医药卫生—放射医学]