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溃疡性结肠炎(ulceractive colitis, UC)属于炎症性肠病的一种,有着较高的发病率,其特征为损伤性炎症,近年来有关其病因及发病机制的研究受到广泛关注,但至今仍不明确[1]。对于溃疡性结肠炎的治疗目前多采用手术、抗感染、糖皮质激素及免疫抑制剂等治疗,但上述治疗手段均为对症治疗,且药物长期使用的不良反应很容易造成疾病复发[2]。因此,探究溃疡性结肠炎的发病机制,将为今后治疗药物的研发提供理论基础。
Metrnl(Meteorin-like)是近年来新发现的神经营养因子,也叫Cometin, Subfatin或是IL-39[3-4]。 Jorgensen等[4]在2012年将Metrnl描述为类似于Meteorin(Metrn)的神经营养因子。Metrnl基因开放阅读框包含4个外显子,由936个碱基对编码311个氨基酸。Metrnl蛋白包含45个N端信号肽序列,切除信号肽后的266个氨基酸构成分子量约为30 000的成熟蛋白分子,整个蛋白分子没有穿膜区域,是一种分泌蛋白。到目前为止,关于Metrnl功能的研究较少,我们前期针对该蛋白相关研究确认Metrnl为一种新的细胞因子,阐明了Metrnl通过PPARγ信号通路介导胰岛素的增敏作用的重要机制[5]。 Jorgensen等[4]报道了Metrnl在神经突触生长和成神经细胞迁移中的神经营养活性。Watanabe等[6]报道,Metrnl是潜伏过程(Latent process,LP)基因,可用于细胞分化和神经突触延伸。脂肪组织Metrnl能促进脂肪细胞分化、改善代谢、抑制炎症从而调节脂肪功能,对抗肥胖引起的胰岛素抵抗[7]。通过检测Metrnl在各种组织中的表达,我们发现Metrnl在人和小鼠胃肠组织中,特别是在肠上皮细胞中都高度表达,并发现肠上皮Metrnl敲除后可以通过抑制肠上皮细胞的自噬而加重溃疡性结肠炎,提示 Metrnl是溃疡性结肠炎的治疗靶点[8]。
肠道微环境形成了良好的微生物群栖息地,肠道微生物群被认为是人体的重要器官,越来越多的研究将这种微生物环境与胃肠道疾病联系起来。肠道菌群在溃疡性结肠炎中起着重要作用,如在无菌状态下,无法制备出某些小鼠结肠炎模型(如IL-10缺陷型小鼠等)[9-10]。也有研究报道,在治疗溃疡性结肠炎患者时,联合使用抗生素也显示出了较好疗效[11]。此外,与健康人群相比,溃疡性结肠炎患者的肠道菌群组成也发生了显著变化[12]。但由于人类肠道菌群的复杂性和多样性,目前尚未清楚某些特定菌属与溃疡性结肠炎发病机制的关系。
因此,本研究聚焦溃疡性结肠炎,从肠道微生态角度出发,探究肠上皮Metrnl对于溃疡性结肠炎的作用以及对肠道菌群调节机制的影响。
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雄性C57小鼠(8周龄,20只,16~20 g),上海西普尔-必凯实验动物有限公司(生产许可证号:SCXK(沪)2013-0016)。Villin-cre小鼠[B6.Cg-Tg(Vil1in-cre)1000Gum/J,021504],2只,16~20 g,美国JAX公司(北京澄天生物科技有限公司代理),生产许可证号:SYXK(京)2018-0016),用于产生肠上皮细胞特异性Metrnl基因敲除小鼠(Metrnl(-/-))。所有小鼠饲养于相对洁净环境下,使用独立通风系统(individual ventilated cages, IVC)动物房,温度恒定(22~26 ℃),室内明暗交替12 h(08:00至20:00照明),相对湿度为40%~70%,笼内维持正压20~25 Pa,每小时换气60~70次。所有实验动物的使用,都经过海军军医大学动物管理机构的同意和认证,符合实验动物饲养及相关管理规定。所有动物实验均按照美国国家卫生研究院实验动物的护理和使用指南进行,并得到海军军医大学动物伦理委员会的批准。IVC系统购自上海鸣励实验室科技发展有限公司。TRIzol试剂(15596026),美国Invitrogen公司;葡聚糖硫酸钠盐(dextran sodium sulfate,DSS),MFCD00081551,分子量36 000~50 000,美国MP公司,引物,生工生物工程有限公司;包埋机(JB- L5,德国徕卡有限公司);切片机(RM2126,德国徕卡有限公司);RT-PCR仪器(ABI 7500系统,美国赛默飞公司);粪便DNA提取试剂盒(QIAamp Fast DNA Stool Mini Kit,Qiagen, Hilden, 德国);紫外微量分光光度计(NanoDrop 2000,Thermo Scientific, 美国);DNA凝胶回收试剂盒(AxyPrep DNA GelExtraction Kit,Axygen Biosciences, 美国);微型荧光计(QuantiFluor-ST,Promega, 美国);测序仪(Illumina MiSeq,Illumina, 美国)。
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首先按照本课题组已报道的方法[5]制备Metrnlloxp/loxp小鼠。根据报道的Metrnl(-/-)小鼠的繁殖策略[13],即将Metrnlloxp/loxp小鼠与购买的Villin-Cre小鼠进行交配,产下后代小鼠基因型为Metrnlloxp/wtVillin-Cre。将Metrnlloxp/wtVillin-Cre小鼠和Metrnlloxp/loxp小鼠交配,产生下一代Metrnlloxp/loxpVillin-Cre小鼠。继续与Metrnlloxp/loxp交配,产下的后代,经基因型鉴定分别为Metrnlloxp/loxpVillin-Cre(Metrnl(-/-))和Metrnlloxp/loxp(Metrnl(+/+))。
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按照本课题组已报道的方法[3],使用TRIzol试剂从肠道组织中提取总RNA,并使用ABI 7500系统进行RT-PCR。最终的20 μl反应混合物包括10 μl SYBR Green,2 μl cDNA模板和1 μl引物。通过重复反应确定平均阈值循环(Ct),将靶基因表达标准化为GAPDH,并使用ΔΔCT方法获得定量测量结果。Metrnl上游引物(F)CTGGAGCAGGGAGGCTTATTT,下游引物(R)GGACAACAAAGTCACTGGTACAG;GAPDH上游引物(F)GTATGACTCCACTCACGGCAAA,下游引物(R)GGTCTCGCTCCTGGAAGATG。
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雄性C57小鼠(8周龄)于实验室适应2周后,按照本课题组已报道的方法进行模型制备[7],将DSS溶于水中,分别至终浓度为3%和1%,让小鼠自由饮用。
小鼠溃疡性结肠炎疾病程度评分,按照我们之前已报道的的评分标准进行评分[8],对体重下降程度、大便性状、血便情况共3部分分别进行评分,然后进行加和,计算总分数。具体评分标准如下(表1)。
表 1 小鼠溃疡性结肠炎疾病程度评分表
疾病评分 体重下降 (%) 大便性状 大便潜血 0 <1 正常 阴性 1 ≥1-5 - + 2 ≥5-10 软 ++ 3 ≥10-15 - +++ 4 ≥15 腹泻 ++++ 注:“-” 无此性状;“+” 潜血程度。 -
小鼠处死后,取整个结肠部位,测量长度进行比较。然后将结肠下段部位组织用4%多聚甲醛固定,石蜡包埋,切片机切至4 μm的切片,按照之前的实验方法[14],进行HE染色,染色后在光学显微镜下观察炎症细胞浸润情况,组织损伤情况并拍照记录。
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使用16S核糖体RNA基因测序技术检测肠道菌群。为了进行样品收集和DNA提取,从实验小鼠中收集粪便样品,并在取样后3h内将其冷冻在−80°C下。使用QIAamp Fast DNA Stool Mini Kit进行DNA提取。使用NanoDrop 2000测量细菌DNA的浓度。然后,将16S核糖体RNA基因测序用于检测细菌DNA。基因的V3-V4区域使用FastPfu聚合酶通过条形码索引引物(338F和806R)进行PCR扩增。然后通过AxyPrep DNA GelExtraction Kit,凝胶提取纯化扩增子,并使用QuantiFluor-ST进行定量。将纯化的扩增子以等摩尔浓度合并,并使用Illumina MiSeq仪器进行末端配对测序。
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16S rRNA测序数据由Quantitative Insights Into Microbial Ecology平台(V.1.9.1)处理,并进行了MegaBLAST搜索,将生物分类单位的读数(OTU)与国家生物技术信息中心16S rRNA数据库中的参考序列比对。按照文献报道的方法[15]进行宏基因组学分析,从16S rRNA序列推算肠道微生物组的基因组,并且对每个样品的基因含量进行了预测。
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本实验结果数据以(
$\bar x $ ±s)表示,使用SPSS18.0软件进行统计分析。多组以上比较采用单因素方差分析(One-way ANOVA),各组与正常对照组比较采用Dunnett t检验法,两组比较采用独立样本t检验。以P<0.05为差异具有统计学意义。
Effect of intestinal Metrnl gene knockout on intestinal microbiota and ulcerative colitis
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摘要:
目的 探讨肠上皮Metrnl对葡聚糖硫酸钠盐(DSS)诱导的溃疡性结肠炎小鼠模型的作用及其对肠道菌群调节机制的影响。 方法 应用不同浓度的DSS(3%和1%)对C57 小鼠进行造模,确定实验条件。给予肠上皮Metrnl特异性敲除小鼠(Metrnl(-/-))及其对照小鼠(Metrnl(+/+))3%DSS造模5 d,观察模型小鼠的生存时间、体重、疾病评分(DAI)、结肠长度以及结肠组织切片病理学变化等指标。使用16S核糖体RNA基因测序技术检测肠道菌群组成。 结果 与1%DSS相比,3% DSS可显著缩短C57小鼠的生存时间(P<0.05),降低体重(P<0.05),增加DAI评分(P<0.05),缩短结肠长度(P<0.05),增加病理学评分(P<0.05)。给予3%DSS造模5 d后,与对照组Metrnl(+/+)小鼠相比,Metrnl(-/-)小鼠体重下降更多(P<0.05),DAI评分更高(P<0.05),结肠长度更短(P<0.05)以及病理学评分更高(P<0.05)。检测16S核糖体RNA结果显示,Metrnl(-/-)小鼠的肠道菌群多样性下降,拟杆菌(Bacteroidetes)和变形杆菌(Proteobacteria)显著降低,而厚壁菌(Firmicutes)显著升高。 结论 Metrnl对3%DSS诱导的溃疡性结肠炎小鼠有保护作用,该作用可能与Metrnl对肠道菌群的调节作用相关。 -
关键词:
- Metrnl基因敲除 /
- 溃疡性结肠炎 /
- 肠道菌群
Abstract:Objective To investigate the effect of intestinal Metrnl on dextran sodium sulfate (DSS)-induced ulcerative colitis mouse model and the regulation mechanism of intestinal microbiota. Methods Different concentrations of DSS (3% DSS and 1% DSS) were used to induce ulcerative colitis on C57 mice to determine the experimental conditions. Intestinal epithelial Metrnl specific knockout mice (Metrnl(-/-)) and its control mice (Metrnl(+/+)) were administrated with 3% DSS for 5 d. Then the survival time, body weight, DAI (disease activity index), colon length and pathological changes in colon tissues were observed. 16S ribosomal RNA gene sequencing was used to detect the composition of intestinal microbiota. Results Compared with 1% DSS, 3% DSS could significantly aggravate ulcerative colitis on C57 mice, such as lower survival rate (P<0.05), more weight loss (P<0.05), higher DAI score (P<0.05), shorter colon length (P<0.05) and higher pathology score (P<0.05). After administrated to 3% DSS for 5 d, comparing with Metrnl(+/+) mice, Metrnl(-/-) mice showed more weight loss (P<0.05), higher DAI score (P<0.05), shorter colon length (P<0.05) and higher pathology score (P<0.05). The 16S ribosomal RNA results showed that the diversity of intestinal microbiota in Metrnl(-/-) mice significantly decreased. Furthermore, Bacteroidetes and Proteobacteria significantly decreased, while Firmicutes increased. Conclusion Metrnl could protect the DSS-induced ulcerative colitis mouse through regulating intestinal microbiota. -
Key words:
- Metrnl gene knockout /
- ulcerative colitis /
- intestinal microbiota
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表 1 小鼠溃疡性结肠炎疾病程度评分表
疾病评分 体重下降 (%) 大便性状 大便潜血 0 <1 正常 阴性 1 ≥1-5 - + 2 ≥5-10 软 ++ 3 ≥10-15 - +++ 4 ≥15 腹泻 ++++ 注:“-” 无此性状;“+” 潜血程度。 -
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