Wang Manxia, Tel: +86-931-5190514; E-mail:
通过建立实验性脑脊髓膜炎(EAE)小鼠模型,观察小鼠肠道菌群在不同发病时间点的变化和炎症因子IL-17、IFN-γ的表达情况,探讨肠道菌群的变化在EAE发病中的免疫调节作用。
将48只C57BL/6小鼠按照随机数字表法分为正常对照组、EAE模型组各24只。EAE组采用MOG35-55与完全弗氏佐剂的混合物制备模型,进行神经功能评分,记录体重变化。分别取免疫后7、14、21、30 d的小鼠粪便,对样本DNA的16S rDNA V3/V4区基因测序。ELASE法检测IL-17、IFN-γ的表达。
EAE组小鼠血液中IL-17、IFN-γ的表达从第7天开始逐渐升高,21 d时达到高峰。14 d时,EAE组与正常对照组相比,物种丰度有显著性差异(
与正常对照组相比,14、21、30 d EAE小鼠肠道菌种的丰度均发生了变化,产生了肠道菌群的紊乱;其中普雷沃氏菌属_
To explore the immune regulatory effect of gut microbiota in experimental autoimmune encephalomyelitis (EAE), we studied the change of gut microbiota and its correlation with the production of IL-17 and IFN-γ at different times.
Female C57BL/6 mice were divided randomly into 2 groups:the normal group and EAE. EAE was induced with MOG35-55 mixed with complete Freund's adjuvant. The weight and neurological scores of 2 groups were observed. The 16S rDNA V3/V4 region of DNA of gut microbiota was indentified by the Illumina Mi Seq high-throughput sequencing.IL-17 and IFN-γ in the blood were detected by ELSIA.
The production of IL-17 and IFN-γ reached the peak on day 21. The abundance of
The abundance and diversity of gut microbiota played the important role at the different times in the EAE group. The production of IL-17 and IFN-γ leaded to the inflammatory of EAE that induced by MOG35-55.
多发性硬化(multiple sclerosis,MS)是中枢神经系统的炎性脱髓鞘性疾病,是一种自身免疫性疾病,具有缓解、复发的临床特点,女性的发病率高于男性,是引起青壮年神经功能障碍最主要的神经系统疾病[
实验性变态反应性脑脊髓炎模型(experimental allergic encephalomyelitis,EAE)是目前公认的MS的理想动物模型,能很好地模拟人类MS的发生、发展和转归过程[
SPF级C57BL/6小鼠48只,雌性,6–8周龄,体重16–18 g,购自甘肃省中医药大学动物研究中心,饲养于甘肃省中医药大学动物实验室(甘肃兰州)。按照随机数字表法分为正常对照组、EAE模型组各24只。已通过兰州大学第二医院伦理委员会审核。
MOG35-55购自CS Bio公司,完全弗氏佐剂(5 mg/mL)购自Sigma公司,结核菌素H37Ra购自Difco公司,百日咳毒素购自LBL公司。酶联免疫法IL-17、IFN-γ试剂盒购自武汉博士德试剂公司。小鼠粪便基因DNA提取试剂盒(QIAGEN,德国)。
0.9%的生理盐水将MOG35-55稀释成10 mg/mL,然后加入等体积的完全弗氏佐剂(CFA)和等量的结核菌素H37Ra,于小鼠脊柱两旁分4点皮下注射0.1 mL的乳剂,之后分别在免疫当天及48 h分2次腹腔注射百日咳毒素0.5 mL作为免疫增强剂。
从免疫后第1天开始,采用双盲法由两名观察者每天称量小鼠体重,采用国际通用的5分评分制对小鼠进行神经功能评分。评分标准:0分:无任何症状;1分:尾部张力消失,可见轻度步态笨拙;2分:一侧后下肢无力,被动翻身后可以恢复;3分:双后肢瘫痪,被动翻身后不能恢复,但给予刺激后可以挪动;4分:双后肢瘫痪伴前肢瘫痪;5分:濒死状态或死亡。
EAE组分别于免疫后7、14、21、30 d留取小鼠粪便,每个时间点3个重复,每个样本0.5 g,取样后迅速至于冻存管中,并迅速放入液氮速冻,之后转移至–80 ℃冰箱保存。
按试剂盒要求提取DNA。PCR扩增基因组DNA,引物为细菌16S rDNA V4区特异性引物,扩增片段长度250 bp,有效序列分布长度构建测序文库采用Illumina MiSeq平台测序,对高质量测序数据进行生物信息学分析,分类学分析采用RDP classifier贝叶斯算法对97%相似性水平的OTU序列进行分类学分析,比对数据库为Silva_12816SrRNA database。EAE 7 d组、14 d组分别与30 d组间菌落结构差异显著性分析采用Metastats软件,7、14、21、30 d多组间比较用LEfSE在线分析软件。
EAE组小鼠分别于后免疫第7、14、21、30天随机各6只小鼠取血,分离血清,酶联免疫法测定IL-17、IFN-γ的含量。具体方法参照试剂盒说明书。
得到的数据采用SPSS 22.0软件,采用重复测量方差分析,计量资料采用(
EAE组小鼠平均发病时间7.70±0.67 d,21 d时达高峰。发病时食欲下降,体重减轻,皮毛不光滑,伴随有神经功能缺损症状出现,起病时尾部张力下降,身体向一侧倾倒,双侧后肢无力,逐渐前肢无力,严重时死亡。EAE组小鼠14 d神经功能评分2.10±1.58,21 d神经功能评分2.93±1.43,两两比较
EAE组小鼠神经功能评分变化趋势图
EAE groups neurological scores in days.
正常对照组、EAE组体重变化趋势图
The weights of normal group and EAE group in days.
ELISA试剂盒检测IL-17、IFN-γ的表达。EAE组血清中IL-17的浓度21 d明显高于14 d和30 d组,差异有统计学意义(
IFN-γ、IL-17的浓度21 d与14 d和30 d相比较
Compared the expression of IFN-γ and IL-17 in 21 days, 14 days and 30 days.
根据16S rRNA测序结果,采用RDP classifier贝叶斯算法对97%相似性水平的OTU序列进行分类学分析,比对数据库Silva_128 16S rDNA database,得到了EAE组和正常对照组粪便共OTU221,15个界,125个门,189个钢,189个目,336个科,688个属,38个种。
采用R语言作图在属水平上TOP 30物种分布柱状图(
TOP30物种分布柱状图
Relative abundance of top 30 (genus level). A: 7 days; B: 14 days; C: 21 days; D: 30 days; E: normal group.
对所有样本进行主坐标分析,图中一个点代表一个样品,颜色相同的点属于同一种分组,两个点之间的距离越近,说明两个样品的微生物群落差异越小(
各样本的主坐标分析
PCoA of samples. A: 7 days; B: 14 days; C: 21 days; D: 30 days; E: normal group.
Metastats差异分析:14 d时,EAE组与正常对照组相比,物种丰度有显著性差异(
组间菌落结构Metastats差异显著性分析结果
Metastats difference analysis. EAE group 14 days
组间菌落结构Metastats差异显著性分析结果
Metastats difference analysis. EAE group 30 days
LEfSE差异分析结果
LEfSE difference analysis. A: 7 days; B: 14 days; C: 21 days; D: 30 days; E: normal group.
普雷沃氏菌属
肠道作为人体最大的微生物定殖场所,肠道菌群参与宿主的多项生理过程,在保护肠道屏障、抑制病原微生物和全身免疫反应的形成中起了重要作用[
目前认为,肠道菌群可能通过调节淋巴细胞分化、炎症因子、代谢产物、破坏血脑屏障等机制参与了MS的发生发展过程[
本研究采用16S rDNA测序,采用R语言作图,各个样本在属水平上TOP 30物种丰度分布柱状图(
本实验主要侧重于炎症因子与肠道菌群在实验性脑脊髓膜炎关系中的作用及其机制进行了相关研究。γ型的IFN、IL-I7是在JAK/STAT1-SOCS3信号通路中抑制免疫的重要因子。肠道菌群结构的紊乱、菌群丰度的减少对EAE小鼠中淋巴细胞的分化、炎症因子表达的影响产生了重要的作用。肠道菌群可能从多个方面参与了MS的复发-缓解的病程,通过本研究发现的在EAE发病过程中起密切相关的肠道菌群,如普雷沃氏菌属
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