沉默信息调节因子6基因敲除模型小鼠肠黏膜形态及转录组学的特征性变化  

Characteristic changes of intestinal mucosal morphology and transcriptomics in silent information regulator 6 gene knockout mice

作　　者：游弋晖 宋春晖 陈曦 姚晓宏 柯俊羽 杜群[1] 宋宁[2] 李燕舞[1] You Yihui;Song Chunhui;Chen Xi;Yao Xiaohong;Ke Junyu;Du Qun;Song Ning;Li Yanwu(Science and Technology Innovation Center,Institute of Spleen and Stomach,Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong Province,China;School of Pharmacy,Henan University of Chinese Medicine,Zhengzhou 450046,Henan Province,China)

机构地区：[1]广州中医药大学科技创新中心,脾胃研究所,广东省广州市510405 [2]河南中医药大学药学院,河南省郑州市450046

出　　处：《中国组织工程研究》2022年第32期5119-5125,共7页Chinese Journal of Tissue Engineering Research

基　　金：广东省教育厅特色创新类项目(2018KTSCX043),项目负责人:李燕舞。

摘　　要：背景:肠黏膜更新是维持机体稳态的关键生理过程,其中隐窝干细胞是肠上皮增殖和分化的驱动力。沉默信息调节因子(silent information regulator,SIRT)参与细胞的基因修复、代谢、能量平衡和寿命的调节,在肠上皮稳态中的研究逐步成为热点。肠类器官由单个隐窝干细胞经3D培养形成,可体外呈现肠上皮更新及受损后修复过程,是研究肠黏膜更新的最新模式。目的:探究SIRT6基因特异性敲除(SIRT6-i/-i)小鼠模型肠黏膜形态、肠类器官形成及其转录组学的特征性变化。方法:①采用Cre-loxp方法建立SIRT6-i/-i小鼠模型,制备小肠组织石蜡切片并进行苏木精-伊红染色,观察其病理变化,免疫组化检测肠干细胞标记物Lgr5和潘氏细胞标记物溶菌酶的表达;②体外构建SIRT6-i/-i小鼠肠类器官模型,Western-blot检测类器官SIRT6蛋白变化,免疫荧光检测肠类器官Lgr5、细胞角蛋白20和黏蛋白2蛋白表达变化;③采用肠组织进行转录组测序,分析该模型小鼠差异基因及相关功能的特征变化。结果与结论:①与野生型对照小鼠肠黏膜形态比较,SIRT6-i/-i小鼠肠黏膜出现绒毛稀疏、变短,隐窝高度变浅,且杯状细胞数目增多,潘氏细胞数目明显减少,Lgr5和溶菌酶的表达明显降低;②SIRT6-i/-i小鼠肠类器官中SIRT6蛋白表达明显降低,培养中出芽率明显减少,且Lgr5表达降低,吸收上皮细胞标记物细胞角蛋白20和杯状细胞标记物黏蛋白2表达水平升高;③转录组结果显示,与野生型对照小鼠比较,模型小鼠肠组织的差异基因有846个,其中上调基因438个,下调基因408个,差异基因在京都基因与基因组百科全书通路富集前5位的分别是化学致癌、药物代谢、视黄酸代谢、亚油酸代谢和类固醇生物合成,显著富集通路中关联的基因主要为Cyp2c29、Cyp2c65、Cyp3a11、Cyp3a25等Cyp450家族的改变;④结果表明,SIRT6缺失会导致肠道干细胞增殖和分化BACKG ROUND:Intestinal mucosal renewal is a key physiological process to maintain body homeostasis,and stem cells at the crypt base are the driving force for intestinal epithelial prolife ration and differentiation.Silent information regulator(SIRT) is involved in the regulation of cell gene repair,metabolism,energy balance,and lifespan.Research on its role in intesti nal epithelial homeostasis has gradually become a hot spot.Intestinal organoids are formed by 3D culture of single crypt stem cells,which can present the intestinal epithelial renewal and repair process after damage in vitro.It is the latest model for studying intestinal mucosal renewal.OBJECTIVE:To elu cidate the characte ristic changes of intestinal mucosal morphology,intestinal organoid formation and its transcriptomics in a SIRT6 genespecific knockout(SIRT6;) mouse model.METHODS:The Cre-loxp method was used to establish the SIRT6;mouse mode.Paraffin sections of small intestine tissue were prepared to observe the pathological changes using hematoxylin-eosin staining,and the expression of intestinal stem cell marker Lgr5 and Paneth cell marker Lyso was detected by immunohistochemistry.The intestinal organoid SIRT6;mouse model was constructed in vitro,SIRT6 protein expression in intestinal organoids was detected by western blot assay,and the protein expression of Lgr5,cytokeratin 20 and mucin 2 in intestinal organoids was detected by immunofluorescence staining.The intesti nal tissue was used for transcriptome sequencing,and the chara cteristic changes of the transcriptome in the mouse model were analyzed.RESULTS AND CONCLUSION:Compared with the control group,SIRT6;mouse intestinal mucosa showed sparse and sho rtened villi,shallower crypts,increased number of goblet cells,significantly decreased number of Paneth cells,and significantly reduced expression of Lgr5 and Lyso.The expression of SIRT6 protein in the SIRT6;mouse organoid was significantly reduced.The budding rate of organoids and the expression of Lgr5 were reduced,while the expression of

关 键 词：SIRT6 基因敲除 肠类器官 肠黏膜 肠道干细胞 转录组 小鼠 

分 类 号：R459.9[医药卫生—治疗学] R329[医药卫生—临床医学]