肿瘤坏死因子-α对小鼠小肠类器官生长、屏障功能和肠道功能细胞的影响  

Effects of Tumor Necrosis Factor-αon Mouse Small Intestinal Organoid Growth,Barrier Function and Intestinal Functional Cells

作　　者：贺文胜 谢文帅 李顺康 匡雁玲 刘玉兰[1] 王丹 HE Wensheng;XIE Wenshuai;LI Shunkang;KUANG Yanling;LIU Yulan;WANG Dan(Hubei Provincial Key Laboratory of Animal Nutrition and Feed Science,Wuhan Polytechnic University,Wuhan 430023,China)

机构地区：[1]武汉轻工大学,动物营养与饲料科学湖北省重点实验室,武汉430023

出　　处：《中国畜牧兽医》2024年第2期491-499,共9页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金青年项目(32102566)。

摘　　要：[目的]研究肿瘤坏死因子-α(TNF-α)对小肠类器官生长、紧密连接蛋白及各种功能细胞标记基因的影响,以建立小肠类器官的疾病损伤模型。[方法]取小鼠小肠,用温和细胞解离试剂(GCDR)消化液分离小鼠隐窝细胞并用肠道类器官培养基培养。选取0(对照组)、50、250、500 ng/mL TNF-α刺激小肠类器官48 h,光学显微镜下观察类器官生长情况,Edu染色示踪细胞增殖的情况,利用实时荧光定量PCR检测细胞增殖、屏障功能和肠道功能细胞标记基因mRNA表达水平。[结果](1)与对照组相比,50和250 ng/mL TNF-α显著降低小肠类器官的出芽率(P<0.05),而对类器官形成率无影响(P>0.05);250和500 ng/mL TNF-α导致小肠类器官坏死率显著升高(P<0.05)。(2)与对照组相比,250 ng/mL TNF-α显著降低小肠类器官紧密连接蛋白Occludin mRNA表达量(P<0.05);500 ng/mL TNF-α显著提高小肠类器官紧密连接蛋白Claudin-1 mRNA表达量(P<0.05)。(3)与对照组相比,50、250、500 ng/mL的TNF-α均导致TNF-α mRNA表达量显著上升(P<0.05),但对白细胞介素6(IL-6)的表达量无显著影响(P>0.05);250和500 ng/mL TNF-α导致IL-1β mRNA表达量显著上升(P<0.05)。(4)250 ng/mL TNF-α导致增殖细胞标记基因Ki67和Pcna基因mRNA表达量显著降低(P<0.05)。(5)与对照组相比,50 ng/mL TNF-α刺激显著降低Lgr5基因mRNA表达量(P<0.05);250和500 ng/mL TNF-α刺激显著降低Muc2、Chga和Lyz基因mRNA表达量;250 ng/mL TNF-α刺激显著降低Alpi基因mRNA表达量(P<0.05)。[结论]250 ng/mL TNF-α刺激可抑制小肠类器官的生长,抑制肠道干细胞的增殖及各种功能细胞的分化。本研究结果可为今后临床应用提供参考。[Objective]The effects of tumor necrosis factor-α(TNF-α)on small intestinal organoid growth,tight junction proteins,and various functional cell marker genes were studied to establish disease damage models for small intestinal organoids.[Method]The crypt cells were isolated from mouse small intestine with gentle cell dissociation reagent(GCDR)digestion solution and cultured in intestinal organoid medium.Small intestinal organoids were stimulated with 0(control group),50,250 and 500 ng/mL TNF-αfor 48 h.The growth of organoids was observed under an optical microscope,and cell proliferation was tracked by Edu staining.The expression levels of cell proliferation,barrier function and intestinal functional cell marker genes mRNA were detected by Real-time fluorescent quantitative PCR.[Result]①Compared with control group,50 and 250 ng/mL TNF-αsignificantly reduced the germination rate of small intestinal organoids(P<0.05),and TNF-αstimulation had no effect on organoid formation rate(P>0.05).250 and 500 ng/mL TNF-αresulted in a significantly higher rate of small intestianl organoid necrosis(P<0.05).②Compared with control group,250 ng/mL TNF-αsignificantly reduced the mRNA expression of the small intestinal organoid tight junction protein Occludin(P<0.05).500 ng/mL TNF-αincreased mRNA expression of Claudin-1(P<0.05),a tight junction protein in small intestines.③Compared with control group,different concentrations of TNF-αled to a significant increase in the expression of mRNA of TNF-α(P<0.05),but had no significant effect on the expression of IL-6(P>0.05).250 and 500 ng/mL TNF-αresulted in a significant increase in mRNA expression of IL-1β(P<0.05).④Compared with control group,250 ng/mL TNF-αled to a significant decrease in mRNA expression of the marker genes Ki 67 and Pcna of proliferating cells(P<0.05).⑤Compared with control group,50 ng/mL TNF-αstimulation significantly reduced the mRNA expression of Lgr 5 gene(P<0.05),250 and 500 ng/mL TNF-αstimulation significantly reduced mRNA expression in Muc

关 键 词：肿瘤坏死因子-α(TNF-α) 小肠类器官 出芽率 紧密连接蛋白 肠道功能细胞 

分 类 号：S864.13[农业科学—野生动物驯养]