文章摘要
丁 菲,夏 凡,狄文娟,丁国宪.雌性11β⁃HSD1敲除小鼠肠道类器官体外研究[J].南京医科大学学报,2019,(5):653~658
雌性11β⁃HSD1敲除小鼠肠道类器官体外研究
In vitro study of intestinal organoids in female 11β⁃HSD1 knockout mice
投稿时间:2018-12-13  
DOI:10.7655/NYDXBNS20190505
中文关键词: 11β⁃HSD1  肠道  肠上皮  类器官  迷你肠  干细胞  潘氏细胞
英文关键词: 11β⁃HSD1  intestine  intestinal epithelium  organoids  mini⁃gut  stem cells  Paneth cells
基金项目:国家自然科学基金项目(81370950)
作者单位
丁 菲 南京医科大学第一附属医院老年内分泌科江苏 南京 210029 
夏 凡 南京医科大学第一附属医院老年内分泌科江苏 南京 210029 
狄文娟 南京医科大学第一附属医院老年内分泌科江苏 南京 210029 
丁国宪 南京医科大学第一附属医院老年内分泌科江苏 南京 210029 
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中文摘要:
      目的:研究雌性11β?HSD1敲除小鼠肠道类器官功能改变。方法:分离年龄性别匹配的C57BL/6J小鼠和11β?HSD1敲除小鼠的肠道组织。细胞流式定量分析肠道上皮层干细胞、祖细胞、潘氏细胞分别所占比例。实时定量PCR检测干细胞和潘氏细胞的标志基因表达情况。分离小鼠肠上皮隐窝单位进行体外3D类器官培养,观察类器官增殖率及类器官分化程度。免疫荧光染色研究小肠类器官干细胞及潘氏细胞的定位和定量。结果:11β?HSD1敲除小鼠与野生组相比,其小肠上皮所含潘氏细胞数显著增高,干细胞数目无明显差异,祖细胞数目增多,肠道干细胞标志基因Lgr5在小肠上皮的表达有增高趋势。同时在大肠上皮组织中有类似的结果,11β?HSD1敲除组大肠干细胞、祖细胞数目及Lgr5基因表达均增高。体外研究结果显示,小肠隐窝成类器官比例有增高趋势,11β?HSD1敲除组增殖分化出的类器官对比野生组具有更复杂的结构及更多数目的类隐窝区域,但大肠隐窝类器官培养结果未见显著差异。对小肠类器官的干细胞及潘氏细胞染色发现,11β?HSD1敲除小鼠肠道类器官含有更多的潘氏细胞,干细胞数目亦有增多趋势。结论:11β?HSD1敲除后小鼠肠道细胞组成发生改变,在小肠上皮组织中,潘氏细胞数目显著增多;在大肠上皮组织中,干细胞、祖细胞数目及干细胞标志基因表达量均显著增加。体外研究显示,11β?HSD1敲除组小肠类器官包含更多的潘氏细胞,类器官具有更强的增殖及分化能力。
英文摘要:
      Objective:To study the changes of intestinal organoids function in female 11β?HSD1 knockout mice. Methods:The intestinal tissues of age?sex matched C57BL/6J mice and 11β?HSD1 knockout mice were collected. Fluorescence?activated cell sorting analysis and quantifications of the intestinal epithelial stem cells,progenitor cells and Paneth cells. Real?time quantitative PCR was used to detect the expression of marker genes in stem cells and Paneth cells. Crypt units were isolated for primary organoid culture in vitro,and the percentage of organoid per crypt ratio and the number of crypt?domains per organoid were observed. Immunofluorescence staining was performed to study the localization and quantification of stem cells and Paneth cells among organoids. Results:Compared with the control group,the number of Paneth cells in the small intestine epithelium of the 11β?HSD1 knockout mice was significantly increased,the number of stem cells was not significantly different,the number of progenitor cells was increased,and the expression of the intestinal stem cell marker gene Lgr5 in the intestinal epithelium was elevated. Similar results were observed in the epithelium of the large intestine. The number of large intestine stem cells,progenitor cells and gene Lgr5 expression were increased in the 11β?HSD1 knockout group. In vitro studies showed that the organoid per crypts ratio was increased,and the organoids derived from the 11β?HSD1 knockout group proliferated and differentiated into more complex structures and consist a greater number of crypt?like domains than the wild group. However,there was no significant difference in the results of colonic crypt organ culture. Immunofluorescence staining of stem cells and Paneth cells of small intestinal organs revealed that the intestinal organoids of 11β?HSD1 knockout mice contained more Paneth cells,and the number of stem cells also increased although not significant. Conclusions:The composition of intestinal epithelium changed after 11β?HSD1 mutation. In the small intestinal epithelium,the number of Paneth cells increased significantly. In the large intestinal epithelium,the number of stem cells,progenitor cells and stem cell marker genes increased significantly. In vitro studies have shown that the 11β?HSD1 knockout group contains more Paneth cells,which have more robust proliferation and differentiation ability.
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