正常小鼠小肠类器官体外培养体系的建立与功能鉴定

Establishment and functional identification of in vitro culture system of small intestine organoid in normal mice

目的建立正常小鼠小肠类器官体外培养体系并进行功能鉴定,为慢性肾脏病下肠道的物质转运提供体外研究工具。方法分离提取C57BL/6J小鼠小肠隐窝,于体外三维培养体系中培养,倒置显微镜下观察小肠类器官的形成过程,苏木精-伊红染色观察小肠类器官的组织结构,免疫荧光鉴定小肠类器官的细胞构成,实时荧光定量PCR检测小肠类器官中物质吸收相关转运体的表达。结果成功提取小肠隐窝,并顺利构建小肠及不同肠段类器官,所培养的类器官具有旺盛的增殖能力,传代后仍能维持增殖状态。免疫荧光结果显示小肠类器官表达黏蛋白2、嗜铬粒蛋白A、Oflm4及溶菌酶不同类型肠道细胞标志物。实时荧光定量PCR结果显示小肠类器官表达钙盐、磷酸盐、钠盐吸收相关转运体,且不同肠段类器官钠盐及磷酸盐吸收主要转运体mRNA表达水平与体内一致,符合小肠节段性吸收的特点。结论成功构建小肠及不同肠段类器官,首次观察到类器官中物质吸收相关转运体的表达,为慢性肾脏病下开展肠道物质转运体外研究提供了稳定和便利的工具。

Objective To establish an in vitro culture system of small intestinal organoid in normal mice and perform functional identification,and to provide an in vitro research tool for material transport in the intestine under chronic kidney disease.Methods The small intestinal crypts of C57BL/6J mice were isolated,extracted and cultured in an in vitro three dimension culture system.The formation of small intestinal organoid was observed with inverted microscope.The tissue structure of the small intestinal organoid was observed by hematoxylin and eosin staining.The cellular composition of the small intestinal organoid was identified by immunofluorescence.The expression of substance absorption⁃related transporters in the small intestinal organoid was detected by real time fluorescence quantitative PCR.Results The small intestinal crypts were successfully extracted.The organoids of small intestine and different intestinal segments were successfully constructed.The cultured organoids had vigorous proliferation ability and maintained proliferation ability after passing through generations.Immunofluorescence results showed that the small intestinal organoids expressed mucin2,chromogranin A,oflm4 and lysozyme,which were different types of intestinal cell biomarkers.The PCR results showed that small intestinal organoids expressed calcium,phosphate and sodium absorption⁃related transporters,and the mRNA expression levels of major transporters for sodium and phosphate absorption in different intestinal segments⁃like organs were consistent with those in vivo,which was consistent with the characteristics of small intestinal segmental absorption.Conclusions The successful construction of small intestine and different intestinal segments organoids,and the first observation of the expression of substance absorption⁃related transporters in such organoid,provide a stable and convenient in vitro research tool for the development of intestinal substance transport in chronic kidney disease.