小鼠肠上皮细胞岩藻糖基化与新生儿坏死性小肠结肠炎发生的关系
|
摘要
目的观察肠上皮细胞岩藻糖基化水平在新生儿坏死性小肠结肠炎(NEC)小鼠模型中的变化,探讨其可能的机制。方法将42只10日龄的C57BL/6新生小鼠按随机数字表法分为NEC组(n=21,采用人工喂养+缺氧+冷刺激方法建立NEC模型)和对照组(n=21,母鼠喂养,不做处理),建模3d后处死。采用NEC病理损伤评分评估建模效果;采用流式细胞技术检测岩藻糖基化肠上皮细胞(F-ECs)和肠道固有层3型天然淋巴细胞(ILC3s)比例;采用实时荧光定量PCR检测肠上皮细胞白细胞介素-22受体(IL-22R)、岩藻糖基转移酶2(Fut2)和固有层淋巴细胞白细胞介素-22(IL-22)的表达水平;采用ELISA法检测固有层淋巴细胞IL-22蛋白的表达水平。结果成功建立NEC小鼠模型。流式细胞检测结果显示,NEC模型组F-ECs百分比低于对照组,差异有统计学意义(P<0.05);NEC模型组肠道固有层ILC3s百分比也明显低于对照组,差异有统计学意义(P<0.001)。实时荧光定量PCR结果显示,NEC模型组上皮细胞IL-22R、Fut2 mRNA表达水平明显低于对照组(P<0.001),固有层淋巴细胞IL-22 mRNA表达水平也明显低于对照组,差异有统计学意义(P<0.01)。NEC模型组固有层淋巴细胞IL-22蛋白表达水平低于对照组,差异有统计学意义(P<0.001)。结论肠上皮细胞岩藻糖基化参与了NEC小鼠模型的发病,其机制可能与ILC3s-IL-22-Fut2轴有关。
Objective To investigate the changes of fucosylation level of intestinal epithelial cells in the mouse model of neonatal necrotizing enterocolitis(NEC), and explore it's possible mechanism. Methods Forty-two 10-day old C57 BL/6 mice were randomly divided into NEC group(n=21, NEC model was established by artificial feeding, hypoxia and cold stimulation) and control group(n=21, mother feeding and no treatment), and mice were sacrificed 3 days after modeling. NEC pathological lesion scoring was performed to assess the modeling effect. Flow cytometry was used to detect the proportion of fucosylated intestinal epithelial cells(F-ECs) and intestinal lamina propria group 3 innate lymphocytes(ILC3 s). The levels of interleukin-22 receptor(IL-22 R), fucosyltransferase 2(Fut2) of intestinal epithelial cells and interleukin-22(IL-22) of lamina propria lymphocyte were detected by quantitative real-time PCR. The level of IL-22 protein in lamina propria was detected by enzyme-linked immunosorbent assay(ELISA). Results The NEC mouse model was successfully established. Flow cytometry results showed that the percentage of F-ECs was lower in NEC model group than in control group(P<0.05). The percentage of intestinal ILC3 s was also significantly lower in NEC model group than in control group with statistically significant difference(P<0.001). The results of quantitative real-time PCR showed that the mRNA levels of IL-22 R and Fut2 were significantly lower in epithelial cells of NEC model group than in control group(P<0.001). The mRNA level of IL-22 was also significantly lower in lamina propria lymphocytes of NEC model group than in control group(P<0.01). The expression level of IL-22 protein in lamina propria lymphocytes was lower in NEC model group than in control group(P<0.001). Conclusion Decreased fucosylation of intestinal epithelial cells is involved in the pathogenesis of NEC mouse model, and the mechanism may be related to the ILC3 s-IL-22-Fut2 axis.
Objective To investigate the changes of fucosylation level of intestinal epithelial cells in the mouse model of neonatal necrotizing enterocolitis(NEC), and explore it's possible mechanism. Methods Forty-two 10-day old C57 BL/6 mice were randomly divided into NEC group(n=21, NEC model was established by artificial feeding, hypoxia and cold stimulation) and control group(n=21, mother feeding and no treatment), and mice were sacrificed 3 days after modeling. NEC pathological lesion scoring was performed to assess the modeling effect. Flow cytometry was used to detect the proportion of fucosylated intestinal epithelial cells(F-ECs) and intestinal lamina propria group 3 innate lymphocytes(ILC3 s). The levels of interleukin-22 receptor(IL-22 R), fucosyltransferase 2(Fut2) of intestinal epithelial cells and interleukin-22(IL-22) of lamina propria lymphocyte were detected by quantitative real-time PCR. The level of IL-22 protein in lamina propria was detected by enzyme-linked immunosorbent assay(ELISA). Results The NEC mouse model was successfully established. Flow cytometry results showed that the percentage of F-ECs was lower in NEC model group than in control group(P<0.05). The percentage of intestinal ILC3 s was also significantly lower in NEC model group than in control group with statistically significant difference(P<0.001). The results of quantitative real-time PCR showed that the mRNA levels of IL-22 R and Fut2 were significantly lower in epithelial cells of NEC model group than in control group(P<0.001). The mRNA level of IL-22 was also significantly lower in lamina propria lymphocytes of NEC model group than in control group(P<0.01). The expression level of IL-22 protein in lamina propria lymphocytes was lower in NEC model group than in control group(P<0.001). Conclusion Decreased fucosylation of intestinal epithelial cells is involved in the pathogenesis of NEC mouse model, and the mechanism may be related to the ILC3 s-IL-22-Fut2 axis.
引文
[1]Tian JY,Zhu T,Wang J,et al.Analysis on association between polymorphism of CD14 and IL-8 gene and susceptibility of necrotizing enterocolitis[J].J Jilin Univ(Med Ed),2016,42(5):958-962.[田佳怡,朱彤,王健,等.CD14和IL-8基因多态性与新生儿坏死性小肠结肠炎易感性的关联性分析[J].吉林大学学报(医学版),2016,42(5):958-962.]
[2]Neu J,Walker WA.Necrotizing enterocolitis[J].N Engl J Med,2011,364(3):255-264.
[3]Shao XM,Ye HM,Qiu XS.Practical neonatology[M].4th Edition.Beijing:People's Medical Publishing House,2011.477-483.[邵肖梅,叶鸿瑁,丘小汕.实用新生儿学[M].4版.北京:人民卫生出版社,2011.477-483.]
[4]Patel RM,Denning PW.Intestinal microbiota and its relationship with necrotizing enterocolitis[J].Pediatr Res,2015,78(3):232-238.
[5]Elgin TG,Kern SL,Mcelroy SJ.Development of the neonatal intestinal microbiome and its association with necrotizing enterocolitis[J].Clin Ther,2016,38(4):706-715.
[6]Xiao GZ,Li J,Yi WH,et al.Influence of glutamine on barrier function of heat-stressed intestinal epithelial Caco-2 cells[J].Med J Chin PLA,2017,42(6):506-510.[肖桂珍,李俊,易万华,等.谷氨酰胺对热打击下单层肠上皮Caco-2细胞屏障功能的影响[J].解放军医学杂志,2017,42(6):506-510.]
[7]Li ZG,Jiang HY,Li LJ,et al.Expression of Gal-9 in peripheral blood and colonic epithelial tissue of mice with colitis[J].JZhengzhou Univ(Med Sci),2015,50(1):68-71.[李治国,江红艳,李林静,等.结肠炎小鼠外周血和结肠上皮组织中Gal-9的表达[J].郑州大学学报(医学版),2015,50(1):68-71.]
[8]Goto Y,Uematsu S,Kiyono H.Epithelial glycosylation in gut homeostasis and inflammation[J].Nat Immunol,2016,17(11):1244-1251.
[9]Pham TA,Clare S,Goulding D,et al.Epithelial IL-22RA1-mediated fucosylation promotes intestinal colonization resistance to an opportunistic pathogen[J].Cell Host Microbe,2014,16(4):504-516.
[10]Goto Y,Obata T,Kunisawa J,et al.Innate lymphoid cells regulate intestinal epithelial cell glycosylation[J].Science,2014,345(6202):1254009.
[11]Martin NA,Mount Patrick SK,Estrada TE,et al.Active transport of bile acids decreases mucin 2 in neonatal ileum:implications for development of necrotizing enterocolitis[J].PLoS One,2011,6(12):e27191.
[12]Wu SF,Chiu HY,Chen AC,et al.Efficacy of different probiotic combinations on death and necrotizing enterocolitis in a premature rat model[J].J Pediatr Gastroenterol Nutr,2013,57(1):23-28.
[13]Hackam DJ,Upperman JS,Grishin A,et al.Disordered enterocyte signaling and intestinal barrier dysfunction in the pathogenesis of necrotizing enterocolitis[J].Semin Pediatr Surg,2005,14(1):49-57.
[14]Kandasamy J,Huda S,Ambalavanan N,et al.Inflammatory signals that regulate intestinal epithelial renewal,differentiation,migration and cel l death:implications for necrotizing enterocolitis[J].Pathophysiology,2014,21(1):67-80.
[15]Clark JA,Doelle SM,Halpern MD,et al.Intestinal barrier failure during experimental necrotizing enterocolitis:protective effect of EGF treatment[J].Am J Physiol Gastrointest Liver Physiol,2006,291(5):G938-G949.
[16]Warner BB,Deych E,Zhou Y,et al.Gut bacteria dysbiosis and necrotising enterocolitis in very low birthweight infants:a prospective case-control study[J].Lancet,2016,387(10031):1928-1936.
[17]Wang Y,Hoenig JD,Malin KJ,et al.16S rRNA gene-based analysis of fecal microbiota from preterm infants with and without necrotizing enterocolitis[J].ISME J,2009,3(8):944-954.
[18]Torrazza RM,Ukhanova M,Wang X,et al.Intestinal microbial ecology and environmental factors affecting necrotizing enterocolitis[J].PLoS One,2013,8(12):e83304.
[19]Lee YK,Mazmanian SK.Has the microbiota played a critical role in the evolution of the adaptive immune system?[J].Science,2010,330(6012):1768-1773.
[20]Round JL,Mazmanian SK.Inducible Foxp3+regulatory T-cell development by a commensal bacterium of the intestinal microbiota[J].Proc Natl Acad Sci U S A,2010,107(27):12204-12209.
[21]Mazmanian SK,Round JL,Kasper DL.A microbial symbiosis factor prevents intestinal inflammatory disease[J].Nature,2008,453(7195):620-625.
[22]Li J,Hsu HC,Mountz JD,et al.Unmasking fucosylation:from cell adhesion to immune system regulation and diseases[J].Cell Chem Biol,2018,25(5):499-512.
[23]Umesaki Y,Okada Y,Matsumoto S,et al.Segmented filamentous bacter ia are indigenous intestinal bacter ia that activate intraepithelial lymphocytes and induce MHC classⅡmolecules and fucosyl asialo GM1 glycolipids on the small intestinal epithelial cells in the ex-germ-free mouse[J].Microbiol Immunol,1995,39(8):555-562.
[24]Bry L,Falk P G,Midtvedt T,et al.A model of host-microbial interactions in an open mammalian ecosystem[J].Science,1996,273(6):1380-1383.
[25]Pickard JM,Mauri ce CF,Kinnebrew MA,et al.R apid fucosylation of intestinal epithelium sustains host-commensal symbiosis in sickness[J].Nature,2014,514(7524):638-641.
[26]Montaldo E,Juelke K,Romagnani C.Group 3 innate lymphoid cells(ILC3s):Origin,differentiation,and plasticity in humans and mice[J].Eur J Immunol,2015,45(8):2171-2182.
[27]Sonnenberg GF,Fouser LA,Artis D.Border patrol:regulation of immunity,inflammation and tissue homeostasis at barrier surfaces by IL-22[J].Nat Immunol,2011,12(5):383-390.
[28]Gury-Benari M,Thaiss CA,Serafini N,et al.The spectrum and regulatory landscape of intestinal innate lymphoid cells are shaped by the microbiome[J].Cell,2016,166(5):1231-1246.e13.
[2]Neu J,Walker WA.Necrotizing enterocolitis[J].N Engl J Med,2011,364(3):255-264.
[3]Shao XM,Ye HM,Qiu XS.Practical neonatology[M].4th Edition.Beijing:People's Medical Publishing House,2011.477-483.[邵肖梅,叶鸿瑁,丘小汕.实用新生儿学[M].4版.北京:人民卫生出版社,2011.477-483.]
[4]Patel RM,Denning PW.Intestinal microbiota and its relationship with necrotizing enterocolitis[J].Pediatr Res,2015,78(3):232-238.
[5]Elgin TG,Kern SL,Mcelroy SJ.Development of the neonatal intestinal microbiome and its association with necrotizing enterocolitis[J].Clin Ther,2016,38(4):706-715.
[6]Xiao GZ,Li J,Yi WH,et al.Influence of glutamine on barrier function of heat-stressed intestinal epithelial Caco-2 cells[J].Med J Chin PLA,2017,42(6):506-510.[肖桂珍,李俊,易万华,等.谷氨酰胺对热打击下单层肠上皮Caco-2细胞屏障功能的影响[J].解放军医学杂志,2017,42(6):506-510.]
[7]Li ZG,Jiang HY,Li LJ,et al.Expression of Gal-9 in peripheral blood and colonic epithelial tissue of mice with colitis[J].JZhengzhou Univ(Med Sci),2015,50(1):68-71.[李治国,江红艳,李林静,等.结肠炎小鼠外周血和结肠上皮组织中Gal-9的表达[J].郑州大学学报(医学版),2015,50(1):68-71.]
[8]Goto Y,Uematsu S,Kiyono H.Epithelial glycosylation in gut homeostasis and inflammation[J].Nat Immunol,2016,17(11):1244-1251.
[9]Pham TA,Clare S,Goulding D,et al.Epithelial IL-22RA1-mediated fucosylation promotes intestinal colonization resistance to an opportunistic pathogen[J].Cell Host Microbe,2014,16(4):504-516.
[10]Goto Y,Obata T,Kunisawa J,et al.Innate lymphoid cells regulate intestinal epithelial cell glycosylation[J].Science,2014,345(6202):1254009.
[11]Martin NA,Mount Patrick SK,Estrada TE,et al.Active transport of bile acids decreases mucin 2 in neonatal ileum:implications for development of necrotizing enterocolitis[J].PLoS One,2011,6(12):e27191.
[12]Wu SF,Chiu HY,Chen AC,et al.Efficacy of different probiotic combinations on death and necrotizing enterocolitis in a premature rat model[J].J Pediatr Gastroenterol Nutr,2013,57(1):23-28.
[13]Hackam DJ,Upperman JS,Grishin A,et al.Disordered enterocyte signaling and intestinal barrier dysfunction in the pathogenesis of necrotizing enterocolitis[J].Semin Pediatr Surg,2005,14(1):49-57.
[14]Kandasamy J,Huda S,Ambalavanan N,et al.Inflammatory signals that regulate intestinal epithelial renewal,differentiation,migration and cel l death:implications for necrotizing enterocolitis[J].Pathophysiology,2014,21(1):67-80.
[15]Clark JA,Doelle SM,Halpern MD,et al.Intestinal barrier failure during experimental necrotizing enterocolitis:protective effect of EGF treatment[J].Am J Physiol Gastrointest Liver Physiol,2006,291(5):G938-G949.
[16]Warner BB,Deych E,Zhou Y,et al.Gut bacteria dysbiosis and necrotising enterocolitis in very low birthweight infants:a prospective case-control study[J].Lancet,2016,387(10031):1928-1936.
[17]Wang Y,Hoenig JD,Malin KJ,et al.16S rRNA gene-based analysis of fecal microbiota from preterm infants with and without necrotizing enterocolitis[J].ISME J,2009,3(8):944-954.
[18]Torrazza RM,Ukhanova M,Wang X,et al.Intestinal microbial ecology and environmental factors affecting necrotizing enterocolitis[J].PLoS One,2013,8(12):e83304.
[19]Lee YK,Mazmanian SK.Has the microbiota played a critical role in the evolution of the adaptive immune system?[J].Science,2010,330(6012):1768-1773.
[20]Round JL,Mazmanian SK.Inducible Foxp3+regulatory T-cell development by a commensal bacterium of the intestinal microbiota[J].Proc Natl Acad Sci U S A,2010,107(27):12204-12209.
[21]Mazmanian SK,Round JL,Kasper DL.A microbial symbiosis factor prevents intestinal inflammatory disease[J].Nature,2008,453(7195):620-625.
[22]Li J,Hsu HC,Mountz JD,et al.Unmasking fucosylation:from cell adhesion to immune system regulation and diseases[J].Cell Chem Biol,2018,25(5):499-512.
[23]Umesaki Y,Okada Y,Matsumoto S,et al.Segmented filamentous bacter ia are indigenous intestinal bacter ia that activate intraepithelial lymphocytes and induce MHC classⅡmolecules and fucosyl asialo GM1 glycolipids on the small intestinal epithelial cells in the ex-germ-free mouse[J].Microbiol Immunol,1995,39(8):555-562.
[24]Bry L,Falk P G,Midtvedt T,et al.A model of host-microbial interactions in an open mammalian ecosystem[J].Science,1996,273(6):1380-1383.
[25]Pickard JM,Mauri ce CF,Kinnebrew MA,et al.R apid fucosylation of intestinal epithelium sustains host-commensal symbiosis in sickness[J].Nature,2014,514(7524):638-641.
[26]Montaldo E,Juelke K,Romagnani C.Group 3 innate lymphoid cells(ILC3s):Origin,differentiation,and plasticity in humans and mice[J].Eur J Immunol,2015,45(8):2171-2182.
[27]Sonnenberg GF,Fouser LA,Artis D.Border patrol:regulation of immunity,inflammation and tissue homeostasis at barrier surfaces by IL-22[J].Nat Immunol,2011,12(5):383-390.
[28]Gury-Benari M,Thaiss CA,Serafini N,et al.The spectrum and regulatory landscape of intestinal innate lymphoid cells are shaped by the microbiome[J].Cell,2016,166(5):1231-1246.e13.