川陈皮素在体内外模型中对肠干细胞增殖的影响  

Effects of nobiletin on intestinal stem cell proliferation in vitro and in vivo

作　　者：吴静峰 郭科男[2,3] 张宁[2] 吴天宇 周子默 陆治宇 王锋超[2] 陈文生 WU Jingfeng;GUO Kenan;ZHANG Ning;WU Tianyu;ZHOU Zimo;LU Zhiyu;WANG Fengchao;CHEN Wensheng(Department of Gastroenterology,First Affiliated Hospital,College of Basic Medical Sciences,Army Medical University(Third Military Medical University),Chongqing,400038,China;State Key Laboratory of Trauma and Chemical Poisoning,Institute of Combined Injury,Faculty of Military Preventive Medicine,College of Basic Medical Sciences,Army Medical University(Third Military Medical University),Chongqing,400038,China;Department of Laboratory Animal Sciences,College of Basic Medical Sciences,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]陆军军医大学(第三军医大学)第一附属医院消化内科,重庆400038 [2]陆军军医大学(第三军医大学)军事预防医学系复合伤研究所,创伤与化学中毒全国重点实验室,重庆400038 [3]陆军军医大学(第三军医大学)基础医学院实验动物学教研室,重庆400038

出　　处：《陆军军医大学学报》2023年第21期2195-2205,共11页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(81872556);陆军军医大学创新能力提升项目(2021XJS03);重庆市院士专项(基础研究与前沿探索类:cstc2018jcyj-yszxX0004)。

摘　　要：目的探讨在体内外模型中常用有效剂量川陈皮素(nobiletin,NOB)对肠道干细胞的调节作用。方法构建小鼠结肠癌细胞系MC38的3D培养模型,加入不同浓度NOB,观察克隆死亡和存活。构建小鼠小肠类器官模型,在类器官培养体系中添加不同浓度的NOB,观察类器官的出芽和生长,利用MTT染色后的类器官成像进行面积和吸光值计算。在50、100μmol/L浓度NOB干预体系中不同时间撤掉NOB,观察类器官生长。在50μmol/L浓度NOB处理类器官的体系中分别加入R-spondin1、CHIR99021加强Wnt通路激活信号,观察类器官的生长。对C57/B6J小鼠灌胃不同浓度NOB,连续给药4 d,观察肠道隐窝和绒毛长度比,观察隐窝细胞凋亡,以及隐窝底部干细胞龛中Olfm4/BrdU染色阳性的细胞数量。结果与对照组相比,50μmol/L NOB即可显著促进肿瘤细胞MC38成球后死亡,显著降低克隆形成率(P<0.001)。与对照组相比,NOB从50μmol/L起可以显著抑制肠类器官的出芽,50~200μmol/L NOB可以显著抑制类器官的生长(P<0.001),并且具有剂量依赖效应;NOB撤药后在50μmol/L时肠类器官生长容易恢复,在100μmol/L时难以恢复到正常水平。增强Wnt通路的激活可以部分挽救NOB对肠类器官的抑制作用。在体给予高于常用浓度的NOB不能诱导肠隐窝细胞凋亡,不影响肠干细胞的数目和增殖,不影响隐窝和绒毛比。结论NOB具有抑制正常肠干细胞的能力,可能通过调控干细胞相关通路如Wnt发挥作用,但其在在体不能发挥抑制干细胞增殖的效应;这提示NOB在常规浓度应用的安全性,同时提醒对NOB在在体肿瘤模型中发挥的抑制作用需要进行更深入的评估。Objective To investigate the regulatory effect of nobiletin(NOB)at typical effective doses on intestinal stem cells in vivo and in vitro.Methods After a 3D culture model of mouse colorectal tumor cell line MC38 was constructed,the death and survival of the obtained colonies were observed after treatment of different concentrations of NOB.Mouse small intestinal crypts were cultured in the Matrigel to generate organoids.Different concentrations of NOB were added to the culture system,and the sprouting and growth of the organoids were observed.MTT assay was used to calculate the area and absorbance values of the organoids after staining.The organoid growth was observed by removing 50 and 100μmol/L NOB for different periods.The effects of exogenous R-spondin1 and CHIR99021(activators of Wnt pathway)on the growth of organoids were observed in the culture system with 50 and 100μmol/L NOB treatment.C57/B6J mice were infused with different concentrations of NOB by gavage for 4 consecutive days.The ratio of intestinal crypt to villus length,cell apoptosis,and number of OLfm4 and BrdU double positive cells were calculated and observed.Results Compared with the control group,50μmol/L NOB significantly promoted the death of MC38 cells after sphere formation and significantly reduced the colony formation rate(P<0.001).The dose also significantly inhibited the budding of intestinal organoids,and 50~200μmol/L NOB significantly inhibited the growth of intestinal organoids in a dose-dependent manner when compared with the control group(P<0.001).After NOB withdrawal,the growth of intestinal organoids in the 50μmol/L group was partially recovered,but it was difficult to recover to normal level in the 100μmol/L NOB group.Enhanced Wnt pathway activation could partially rescue the inhibitory effect of NOB on intestinal organoids.In vivo administration of higher concentrations of NOB did not induce apoptosis of intestinal crypt cells,did not affect the crypt to villus ratio,and had no effects on the number and proliferation statu

关 键 词：川陈皮素 小肠干细胞 小肠类器官 细胞增殖 在体 

分 类 号：R285.5[医药卫生—中药学] R322.45[医药卫生—中医学] R329.28