肠道菌群变化对肠上皮细胞Myd88蛋白及炎症因子的影响
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摘要
目的研究肠道菌群变化对体外培养的正常大鼠肠黏膜上皮细胞Myd88蛋白水平及其下游TNF-α等炎症因子的影响。方法选取10只健康大鼠,实验室适应性喂养5d后继续常规喂养7d并处死,取结肠组织进行细胞培养得到原代肠黏膜上皮细胞,将所得细胞分为5组,分别用正常细胞培养液(A组)、正常大鼠肠腔菌溶液(B组)、正常大鼠黏膜菌溶液(C组)、溃疡性结肠炎大鼠肠腔菌溶液(D组)、溃疡性结肠炎大鼠黏膜菌溶液(E组)培养,通过蛋白质印迹法检测各组细胞中Myd88蛋白含量,通过酶联免疫吸附法检测TNF-α和IL-6含量。结果 D组、E组与A组比较,细胞中Myd88蛋白及TNF-α、IL-6水平显著增高(Ps<0.01),D、E组与B、C组比较Myd88蛋白及TNF-α、IL-6水平亦显著增高(Ps<0.01),而B组、C组相较于A组差异无统计学意义(Ps>0.05)。进一步比较D组与E组、B组与C组间Myd88蛋白及TNF-α、IL-6水平差异亦无统计学意义(Ps>0.0.5)。结论 (1)肠道菌群结构的改变可引起肠黏膜细胞Myd88通路的激活及炎症因子的释放。(2)肠道中细菌种类、数量、分布位置等因素的综合变化与肠黏膜炎症反应的发生关系密切。
Objective To observe the impact of intestinal flora variation on the levels of Myd88 protein and inflammatory factors such as TNF-αin normal rat primary colonic epithelial cells cultured in vitro.Methods 10 healthy rats were sacrificed after 5 days of adaptive feeding followed by 7 days of normal feeding in laboratory;the colonic tissue were collected and the primary colonic epithelial cells were cultured in vitro.The cells were randomly divided into 5 groups and cultured with normal cell culture medium(group A),normal rat intestinal cavity bacteria(group B),normal rat intestinal mucosal bacteria(group C),UC rat intestinal cavity bacteria(group D)and UC rat intestinal mucosal bacteria(group E),respectively.Myd88 protein was detected with Western Blot,and TNF-αand IL-6 were detected by using ELLSA.Results The levels of Myd88 protein,TNF-αand IL-6 in group D and E significantly increased than in group A,B and C(Ps<0.01).But,compare with group A,the levels of Myd88 protein,TNF-αand IL-6 in group B and C were not significantly different(Ps>0.0.5).Further more,there were no significant differences(Ps>0.0.5)in the levels of Myd88 and inflammatory factors between group B and C,and between group D and E.Conclusion(1)The variation of intestinal flora structure may activate Myd88 pathway and cause the release of inflammatory factors in intestinal epithelial cells.(2)The changes of bacterial species,quantity and location in intestinal tract have a close relationship with the occurrence of inflammatory reaction of intestinal mucosa.
Objective To observe the impact of intestinal flora variation on the levels of Myd88 protein and inflammatory factors such as TNF-αin normal rat primary colonic epithelial cells cultured in vitro.Methods 10 healthy rats were sacrificed after 5 days of adaptive feeding followed by 7 days of normal feeding in laboratory;the colonic tissue were collected and the primary colonic epithelial cells were cultured in vitro.The cells were randomly divided into 5 groups and cultured with normal cell culture medium(group A),normal rat intestinal cavity bacteria(group B),normal rat intestinal mucosal bacteria(group C),UC rat intestinal cavity bacteria(group D)and UC rat intestinal mucosal bacteria(group E),respectively.Myd88 protein was detected with Western Blot,and TNF-αand IL-6 were detected by using ELLSA.Results The levels of Myd88 protein,TNF-αand IL-6 in group D and E significantly increased than in group A,B and C(Ps<0.01).But,compare with group A,the levels of Myd88 protein,TNF-αand IL-6 in group B and C were not significantly different(Ps>0.0.5).Further more,there were no significant differences(Ps>0.0.5)in the levels of Myd88 and inflammatory factors between group B and C,and between group D and E.Conclusion(1)The variation of intestinal flora structure may activate Myd88 pathway and cause the release of inflammatory factors in intestinal epithelial cells.(2)The changes of bacterial species,quantity and location in intestinal tract have a close relationship with the occurrence of inflammatory reaction of intestinal mucosa.
引文
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[11]LI Runmei,HAN Ying,WANG Jiheng,et al.An analysis of the DNA fingerprinting of intestinal flora in inflammatory bowel disease[J].Chin J Inter Med,2007,46(2):96-98.(in Chinese)李润美,韩英,王继恒,等.炎症性肠病患者肠道菌丛结构特征的指纹图谱分析[J].中华内科杂志,2007,46(2):96-98.
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[13]Qin JJ,Li RQ,Raes JJ,et al.A human gut microbial gene catalogue established by metagenomic sequencing[J].Nature,2010,464(7285):59-65.
[2]WU Ruili,SUN Ziqin,WEI Zhi.Intestinal flora structural features in the ulcerative colitis by ERIC-PCR fingerprinting[J].Chin J Gastroenterol&Hepatol,2012,21(9):798-801.(in Chinese)吴瑞丽,孙自勤,魏志.溃疡性结肠炎肠道菌群结构ERICPCR指纹图谱分析[J].胃肠病学和肝病学杂志,2012,21(9):798-801.
[3]LIU Huan,WANG Lei,GAO Hongliang,et al.Effects of TLR4mAb on the luminal and mucosal microbiotas in rat models of ulcerative colitis[J].Chin J Microecol,2016,28(4):373-377.(in Chinese)刘欢,王磊,高鸿亮,等.TLR4mAb对2,4,6-三硝基苯磺酸诱导的溃疡性结肠炎模型大鼠肠腔及肠黏膜菌群的影响[J].中国微生态学杂志,2016,28(4):373-377.
[4]Evans GS,Flint N,Somers AS,et al.The development of a method for the preparation of rat intestinal epithelial cell primary cultures[J].J Cell Sci,1992,101(1):219-231.
[5]薛庆善.体外培养的原理与技术[M].北京:科学出版社,2000.
[6]Kinnucan JA,Rubin DT,Ali T.Sleep and inflammatory bowel disease:Exploring the relationship between sleep disturbances and inflammation[J].Gastroenterol&Hepatol,2013,9(11):718-727.
[7]Cascio A,Laria C,Ricciardi F,et al.Cytomegalovirus complicating inflammatory bowel disease:Useful remarks[J].Gastroenterol&Hepatol,2013,9(11):756-757.
[8]Egmond M,Vidarsson G,Bakema JE.Cross-talk between pathogen recognizing Toll-like receptors and immunoglobulin Fc receptors in immunity[J].Immunol Rev,2015,268(1):311-327.
[9]Dong L,Li J,Liu Y,et al.Toll-like receptor 2monoclonal antibody or/and Toll-like receptor 4monoclonal antibody increase counts of Lactobacilli and Bifidobacteriain dextran sulfate sodium-induced colitis in mice[J].J Gastroenterol&Hepatol,2012,27(1):110.
[10]Leite FR,de Aquino SG,Guimar2es MR,et al.Relevance of the myeloid differentiation factor 88(MyD88)on rankl,opg,and nod expressions induced by TLR and IL-1Rsignaling in bone marrow stromal cells[J].Inflammation,2015,38(1):1-8.
[11]LI Runmei,HAN Ying,WANG Jiheng,et al.An analysis of the DNA fingerprinting of intestinal flora in inflammatory bowel disease[J].Chin J Inter Med,2007,46(2):96-98.(in Chinese)李润美,韩英,王继恒,等.炎症性肠病患者肠道菌丛结构特征的指纹图谱分析[J].中华内科杂志,2007,46(2):96-98.
[12]Wang SL,Wang ZR,Yang CQ.Meta-analysis of broad-spectrum antibiotic therapy in patients with active inflammatory bowel disease[J].Exp&Therap Med,2012,4(6):1051.
[13]Qin JJ,Li RQ,Raes JJ,et al.A human gut microbial gene catalogue established by metagenomic sequencing[J].Nature,2010,464(7285):59-65.