外源性IL-34对实验性小鼠结肠炎的影响  

Predictive Value of Interleukin-34 for Disease activity in Crohn′s disease and its Protective Effect on Experimental Enteritis in Mice

作　　者：陈冰倩 黄伟[2] 杨君伶[3] 刘肇修[2] CHEN Bing-qian;HUANG Wei;YANG Jun-ling;LIU Zhao-xiu(Affiliated Hospital of Nantong University;Department of Gastroenterology,Affiliated Hospital of Nantong University;Clinical medical Research Center,Affiliated Hospital of Nantong University)

机构地区：[1]南通大学附属医院,226001 [2]南通大学附属医院消化内科,226001 [3]南通大学附属医院临床医学研究中心,226001

出　　处：《现代消化及介入诊疗》2023年第9期1104-1110,共7页Modern Interventional Diagnosis and Treatment in Gastroenterology

基　　金：国家自然科学基金青年基金(82000497);江苏省自然科学基金青年基金(BK20200965);中国博士后科学基金(2019M661909);江苏省博士后支撑基金(2021K405C)。

摘　　要：目的研究人源IL-34重组蛋白对实验性小鼠肠炎的保护作用,初步探究IL-34在肠黏膜屏障中的作用及分子机制。方法1.将20只6~8周C57BL/6J雄性小鼠随机分为两组,每组10只,利用2%葡聚糖硫酸钠(dextran sulphate sodium,DSS)诱导小鼠急性结肠炎模型。其中,实验组分别在DSS造模的第0、1、4、7天腹腔注射rhIL-34蛋白(100 ng/mL),对照组则在相同时间点腹腔注射同剂量的PBS。造模结束后取小鼠全结肠,评估两组小鼠的体重、结肠长度、疾病活动性指数(disease activity index,DAI)及组织病理损伤程度,明确rhIL-34对小鼠急性结肠炎的保护作用;利用蛋白质免疫印迹、免疫组化等实验探究两组小鼠结肠组织中E-cadherin、ZO-1以及Occludin的表达差异,评估rhIL-34对小鼠肠黏膜屏障的影响。2.采用NCM460细胞构建体外细胞模型。其中,实验组采用DSS处理NCM460细胞构建细胞炎症模型,对照组采用同剂量的PBS处理相同时间,qRT-PCR实验检测炎症相关基因的表达,验证造模是否成功;为了进一步探究rhIL-34对细胞的保护作用,在NCM460细胞中提前加入rhIL-34预处理6 h、12 h、24 h(100 ng/mL),后加入DSS诱导细胞炎症模型,24 h后收集细胞,使用蛋白质印迹法、qRT-PCR检测各组细胞中E-cadherin、ZO-1以及Occludin的表达差异。最后利用RNA-Sequencing分析各组样本间的差异表达基因谱。结果1.小鼠肠炎模型结果显示:与对照组相比,rhIL-34腹腔注射组的小鼠体重下降及结肠缩短程度显著减轻、DAI评分下降以及结肠组织损伤程度减弱。并且发现,rhIL-34腹腔注射组小鼠结肠组织中E-cadherin、ZO-1以及Occludin蛋白的表达显著升高。2.体外细胞炎症模型结果显示:rhIL-34提前处理24 h的NCM460细胞中的E-cadherin、ZO-1以及Occludin的表达水平较对照组显著升高。结论rhIL-34可能通过改善肠黏膜屏障,对DSS诱导的实验性小鼠结肠炎起保护作用。Objective To investigate the protective effect of recombinant human IL-34(rhIL-34)protein on experimental mouse enteritis,and to explore the role of IL-34 in intestinal mucosal barrier and its molecular mechanism.Methods 1.Twenty 6-8-week-old C57BL/6J male mice were randomly divided into four groups with 10 mice in each group which included normal drinking water group(control),dextran sulfate sodium salt(DSS)modeling group(DSS),DSS modeling+intraperitoneal injection of PBS group(DSS+PBS),DSS modeling+intraperitoneal injection of Ryl-34 group(DSS+Ryl-34).Mice in DSS+rhIL-34 group were intraperitoneally injected with rhIL-34(100 ng/mL)four times on days 0,1,4 and 7 after DSS modeling.DSS+PBS group was intraperitoneally injected with the same dose of PBS at the same time point.After the modeling,whole colon was taken from mice,and body weight,colon length,disease activity index(DAI)and histopathological injury degree of mice in both groups were assessed,so as to clarify the protective effect of rhIL-34 on acute colitis of mice.The difference of E-cadherin,ZO-1 and Occludin expression in colon tissue of mice in the two groups was investigated by western blotting and immunohistochemistry,and the effect of rhIL-34 on intestinal mucosal barrier of mice was evaluated.2.NCM460 cells were used to construct cell models in vitro.In the experimental group,NCM460 cells were treated with DSS to construct the cell inflammation model,while the control group was treated with PBS at the same dose for the same time.qRT-PCR was used to detect the expression of inflammation-related genes to verify the success of the modeling.In order to further explore the protective effect of rhIL-34 on cells,NCM460 cells were pretreated with rhIL-34 for 6 h,12 h and 24 h(100 ng/mL),then DSS was added to induce cell inflammation model,and cells were collected 24h later.Western blotting and qRT-PCR were used to detect the difference of E-cadherin,ZO-1 and Occludin expression in each group.Finally,RNA Sequencing was used to analyze the differentially e

关 键 词：炎症性肠病、白介素34 疾病活动度 肠黏膜屏障 

分 类 号：R574[医药卫生—消化系统]