摘要
目的探讨巨噬细胞极化在新生儿坏死性小肠结肠炎(necrotizing enterocolitis,NEC)发病过程中的作用。方法选用SPF级C57BL/6小鼠建立NEC小鼠模型,将早产小鼠随机分为对照组(n=10)和NEC组(n=19)。对照组小鼠由母鼠母乳喂养,NEC组小鼠给予缺氧、冷刺激、高渗喂养和脂多糖处理。小鼠出生后96 h收集十二指肠下端至结肠部肠道组织。HE染色观察肠道组织形态;FD70灌胃检测肠道屏障功能;免疫荧光检测肠上皮Pan-keratin表达;原位末端转移酶标记(TUNEL)染色检测肠道上皮细胞凋亡;Western blot检测巨噬细胞极化相关指标CD86和CD206的蛋白表达水平;流式细胞术检测肠道M1型、M2型巨噬细胞比例;RT-PCR检测细胞因子粒细胞-巨噬细胞集落刺激因子、白细胞介素(interleukin,IL)-6、IL-10、IL-12的mRNA表达水平。结果与对照组相比,NEC组小鼠存活率显著降低(100.0%比36.8%),肠道组织出现不同等级损伤,肠上皮完整性遭到破坏,肠上皮细胞凋亡增多(1.9%±1.1%比7.6%±2.6%,P<0.05),肠道通透性增加(1.53±0.80比14.32±1.27,P<0.05)。Western blot结果显示NEC组CD86蛋白表达上升(1.0±0.01比1.5±0.10,P<0.05),CD206蛋白表达下降(1.0±0.01比0.6±0.05,P<0.05);流式细胞学结果显示NEC组CD68^(+)CD86^(+)M1型巨噬细胞比例增加(1.90%±0.19%比10.20%±0.38%,P<0.05),CD68^(+)CD206^(+)M2型巨噬细胞比例减少(5.8%±0.33%比3.7%±0.56%,P<0.05),促炎因子粒细胞-巨噬细胞集落刺激因子、IL-6、IL-12的mRNA(分别为1.00±0.05比1.80±0.17,1.00±0.13比2.00±0.58,和1.00±0.05比1.40±0.22,P<0.05)表达水平明显上升,抗炎因子IL-10的mRNA(1.00±0.22比0.00±0.01,P<0.05)表达水平明显下降。结论巨噬细胞向促炎型M1型极化在NEC的发病过程中起重要作用。
Objective To investigate the effect of macrophage polarization in the pathogenesis of necrotizing enterocolitis(NEC).Methods C57BL/6 mice were chose to construct the NEC model.The preterm pups were randomly assigned into the control group(n=10)and the NEC group(n=19).The pups in the control group were breastfed by mothers while the NEC group were treated with hypoxia,hypothermia,hypertonic feeding and lipopolysaccharide treatment.The intestinal tissues from the lower part of duodenum to the colon were collected after the pups were born after 96 hours.HE staining was used to observe the intestinal histological structure.Intestinal mucosal permeability was detected by the measurement of concentration of FD70 in plasma after gastric gavage.The expression of Pan-keratin of intestinal epithelium was detected by immunofluorescence histochemistry.Enterocyte apoptosis was detected by TUNEL staining.The expression of CD86 and CD206 protein were determined by western blotting and the percentage of M1 and M2 macrophages was calculated by flow cytometry.RT-PCR was used to detect the expression of mRNA levels of granulocyte-macrophage colony stimulating factor,interleukin(IL)-6,IL-10 and IL-12.Results Compared with the control group,the pups in the NEC group had low survival rate(100.0%vs.36.8%),different level of intestinal injury,incomplete integrity of intestinal epithelium,increased mucosal permeability(1.53±0.80 vs.14.32±1.27,P<0.05)and enterocyte apoptosis(1.9%±1.1%vs.7.6%±2.6%,P<0.05).Western blotting showed that the expression of CD86 protein(1.00±0.01 vs.1.50±0.10,P<0.05)increased while CD206 protein decreased(1.00±0.01 vs.0.60±0.05,P<0.05)in the NEC group.Flow cytometry showed that the percentage of CD68^(+)CD86^(+)M1 macrophages increased(1.90%±0.19%vs.10.20%±0.38%,P<0.05)while the CD68^(+)CD206^(+)M2 macrophages decreased(5.8%±0.33%vs.3.7%±0.56%,P<0.05)in the NEC group.The expression of the mRNA levels of pro-inflammatory cytokines,granulocyte-macrophage colony stimulating factor(1.00±0.05 vs.1.83±0.
作者
陶佳鑫
毛靖
苏艳伟
Tao Jiaxin;Mao Jing;Su Yanwei(School of Nursing,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China)
出处
《中国小儿急救医学》
CAS
2021年第12期1047-1053,共7页
Chinese Pediatric Emergency Medicine
基金
湖北省自然科学基金(2019CFB516)
国家自然科学基金(81601330)。
关键词
坏死性小肠结肠炎
巨噬细胞极化促炎因子
抗炎因子
Necrotizing enterocolitis
Macrophage polarization
Pro-inflammatory cytokine
Anti-inflammatory cytokine
