依托泊苷诱导的人小肠类器官衰老模型的构建及鉴定  

Construction and identification of a human small intestinal organoid aging model induced by etoposide

作　　者：武凤云 王应归 肖春 李玉芳 金蕊 程龙 牛畅[1] WU Feng-yun;WANG Ying-gui;XIAO Chun;LI Yu-fang;JIN Rui;CHENG Long;NIU Chang(College of Life Sciences,Capital Normal University,Beijing 100048,China;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization,Ministry of Education,Southwest Minzu University,Chengdu 610225,China;Department of General Surgery,the PLA Rocket Force Characteristic Medical Center,Beijing 100088,China;Institute of Biotechnology,Academy of Military Medical Sciences,Beijing 100089,China)

机构地区：[1]首都师范大学生命科学学院,北京100048 [2]西南民族大学青藏高原动物遗传资源保护与利用四川省/教育部重点实验室,四川成都610225 [3]火箭军特色医学中心普通外科,北京100088 [4]军事科学院军事医学研究院生物工程研究所,北京100089

出　　处：《中国药理学与毒理学杂志》2023年第3期191-196,共6页Chinese Journal of Pharmacology and Toxicology

基　　金：北京市自然科学基金(5182002);国家自然科学基金(82072717);北京市新星交叉课题(Z191100001119020);北京市属高校高水平教师队伍建设支持计划青年拔尖人才培育计划(CIT&TCD201904077);北京市自然科学基金面上专项(M23003)。

摘　　要：目的 用依托泊苷(etoposide)诱导类器官发生DNA损伤构建人小肠类器官衰老模型。方法 临床活检小肠组织,体外无菌分离获得小肠隐窝,在培养基中培养成为类器官。类器官用依托泊苷10μmol·L^(-1)处理7 d,倒置相差显微镜观察类器官表面形态变化,Western印迹法检测DNA损伤标志物磷酸化组蛋白H2AX(γH2AX)水平;衰老相关β半乳糖苷酶(SA-β-gal)活性染色检测类器官SA-β-gal阳性细胞面积百分率,Western印迹法检测衰老标志物p16^(INK4A)蛋白表达水平。结果 人小肠隐窝体外成功培养成球。依托泊苷诱导的人小肠类器官衰老模型表面皱缩,类器官部分死亡,整体表面积变小;类器官中γH2AX蛋白表达显著上调(P<0.01);SA-β-gal阳性细胞面积百分率显著增加(P<0.01),约90%细胞呈SA-β-gal阳性;p16^(INK4A)蛋白表达显著增加(P<0.01)。结论 依托泊苷处理可诱导人小肠类器官发生DNA损伤,从而诱导人小肠类器官衰老。OBJECTIVE To construct human small intestinal organoids and an aging model of human small intestinal organoids.METHODS Small intestinal crypts were isolated by human biopsy small intestinal tissue in vitro and cultured into organoids in culture medium.Small intestinal organoids were treated with etoposide 10μmol·L-1 for 7 d.The surface morphological changes of organoids were observed under a microscope.Phosphorylated histone H2AX(γH2AX)and p16INK4A were detected by Western blotting.Senescence-associatedβ-galactosidase(SA-β-gal)staining was used to detect the positive cell area rate of organoids staining after etoposide treatment.RESULTS Human small intestinal crypts were cultured in vitro before their aging model was established.Compared with the control group,the surface of human small intestinal organoids treated with etoposide contracted.Some of the organoids treated with etoposide died,and overall surface became smaller.Compared with the control group,the expression ofγH2AX in human small intestinal organoids treated with etoposide was significantly up-regulated(P<0.01).Compared with the normal control group,the proportion of the SA-β-gal positive cell area increased significantly(P<0.01),about 90%of the cells were SA-β-gal positive,and the expression of p16INK4A increased significantly(P<0.01).CONCLUSION Etoposide treatment can induce DNA damage to human small intestinal organoids,thereby inducing of senescence human small intestinal organoids.

关 键 词：人小肠类器官 依托泊苷 衰老相关β半乳糖苷酶 p16^(INK4A)A 

分 类 号：R965.1[医药卫生—药理学]