CN113403212B - 一种肠道真菌Candida metapsilosis M2006B及其应用 - Google Patents
一种肠道真菌Candida metapsilosis M2006B及其应用 Download PDFInfo
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Abstract
本发明公开一种肠道真菌Candida metapsilosis M2006B及其应用，涉及微生物领域；所述Candida metapsilosis M2006B的菌种保藏号为GDMCC 61628；本发明进一步结合炎性肠病动物模型评价Candida metapsilosis M2006B的生物活性发现，Candidametapsilosis M2006B能够通过抑制结肠病变，抑制结肠缩短，下调IL‑1β、IL‑6、TNF‑α、INF‑γ等炎症因子表达，对炎性肠病进行预防或治疗；本发明不仅明确了该菌在炎性肠病中的重要作用，还揭示了该菌在炎性肠病防治中的广阔应用前景。
Description
技术领域
本发明涉及微生物领域，特别是涉及一种肠道真菌Candida metapsilosisM2006B及其应用。
背景技术
炎症性肠病(Inflammatory Bowel Disease，IBD)是一类以回肠、直肠、结肠炎症为特征的疾病，主要包括溃疡性结肠炎(Ulcerative Colitis，UC)和克罗恩病亚型(Crohn's Disease，CD)。临床表现主要为持续腹泻、腹痛、直肠出血/便血、体重减轻等，严重影响患者的生活质量，且易复发，被认为是一种终身性疾病。目前，IBD已成为一种全球性疾病，特别是发达国家的发病率显著高于发展中国家，尚无明显的改善趋势。随着人民饮食结构、生活方式和生活环境的显著改变，我国IBD的发病率(1.8/10万人)呈持续快速增长的趋势，已成为消化系统常见疾病。受到炎症性肠病侵害的患者可以及时合理使用美沙拉嗪、泼尼松龙、英夫利昔单抗等药物来治疗疾病。对于难治性炎性肠病，在药物治疗无效的情况下，粪菌移植是一种较为前沿，且行之有效的治疗方法。近年来，以益生菌为主的生物治疗方案日益受到重视。从人肠道中分离鉴定益生菌，是防治炎性肠病的一种有效途径。
肠道中寄生了大量微生物，包括细菌、真菌等，与机体的生理、病理过程密切相关。Candida metapsilosis M2006B作为一种从人粪便中分离鉴定的真菌，其生物功能，特别是对于炎性肠病的作用未见明确报道。因此，本发明综合运用微生物学、药理学实验技术从人源肠道真菌中发现能够用于防治炎性肠病的益生菌资源。
发明内容
本发明的目的是提供一种肠道真菌Candida metapsilosis M2006B及其应用，以解决上述现有技术存在的问题。
为实现上述目的，本发明提供一种肠道真菌Candida metapsilosis M2006B，所述真菌Candida metapsilosis M2006B的菌种保藏号为GDMCC 61628。
本发明还提供一种所述真菌Candida metapsilosis M2006B在制备预防和/或治疗炎症性肠病的产品中的应用。
进一步的，所述炎症性肠病为溃疡性结肠炎或克罗恩病。
进一步的，所述产品为药物、功能食品或添加剂。
进一步的，所述产品中还包括药学上可接受的载体。
进一步的，所述药学上可接受的载体为奶粉、乳糖、环糊精、麦芽糖、葡萄糖、甘油、谷氨酸钠、维生素C、甘露糖、半乳糖、甘露聚醇或甲基纤维素。
进一步的，所述肠道真菌Candida metapsilosis M2006B的有效剂量为5×109CFU/Kg。
本发明公开了以下技术效果：本发明从人粪便样本中分离鉴定一种肠道真菌，鉴定为Candida metapsilosis M2006B。本发明进一步结合炎性肠病动物模型评价其生物活性发现，Candida metapsilosis M2006B能够通过抑制结肠病变，抑制结肠缩短，下调IL-1β、IL-6、TNF-α、INF-γ等炎症因子表达，对炎性肠病进行预防或治疗；本发明不仅明确了Candida metapsilosis M2006B在炎性肠病中的重要作用，还揭示了该菌在炎性肠病防治中的广阔应用前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为Candida metapsilosis M2006B的扫描电镜图；
图2为实施例2中Candida metapsilosis M2006B与对照组相比，对野生型小鼠炎性肠病表型的缓解治疗作用结果图，其中A为小鼠体重变化；B为小鼠腹泻评分；C为小鼠直肠出血或血便情况；
图3为实施例2中Candida metapsilosis M2006B对野生型炎性肠病小鼠结肠病变的影响结果；其中，A为肠镜图，B为结肠组织HE 染色切片图；
图4为实施例2中Candida metapsilosis M2006B对野生型炎性肠病小鼠结肠组织长度的影响；
图5为实施例2中Candida metapsilosis M2006B定植后小鼠结肠组织中炎症因子IL-1β、IL-6、TNF-α、INF-γ的表达情况；
图6为实施例3中Candida metapsilosis M2006B定植后小鼠的体重、活动指数、腹泻评分以及直肠出血或血便情况，其中A为小鼠体重变化；B为小鼠腹泻评分；C为小鼠直肠出血或血便情况；
图7为实施例3中Candida metapsilosis M2006B定植后小鼠的结肠长度结果，其中，A为结肠照片，B为数据统计；
图8为实施例3中Candida metapsilosis M2006B定植后小鼠的结肠组织HE染色切片图；
图9为实施例3中Candida metapsilosis M2006B定植后小鼠结肠组织中炎症因子IL-1β、IL-6、TNF-α、INF-γ的mRNA表达情况。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图和具体实施方式对本发明作进一步详细的说明。
实施例1菌株分离与鉴定
于洁净环境中，取新鲜人粪便样本，经无菌水稀释后，涂布于改良马丁琼脂培养基(MTB，含青霉素100U/mL、链霉素100μg/mL)，于32℃恒温培养5-10天。观察到显著真菌菌落时，挑取形态差异菌落，接种于新配制的MTB培养基上，继续于32℃恒温培养3天，观察到单克隆菌落时，挑取单克隆菌落进一步接种于新配制的MTB培养基上，于32℃恒温培养3天，获得纯化的肠道真菌，为白色圆型菌落，表面光滑平整(编号M2006B)，挑取菌体置于25％甘油水溶液中，于-80℃保存菌种。同时，通过DNA提取试剂盒，对分离菌株的核酸进行提取，通过18S rDNA引物进行RT-PCR，结合测序技术，对分离菌株进行18S rRNA基因测序分析，结合NCBI核酸数据库BLAST比对，确定其种属Candida metapsilosis(同源性99.4％)，图1为Candida metapsilosis M2006B的扫描电镜图。所述念珠菌属真菌Candida metapsilosisM2006B的菌种于2021年4月26日保藏于广东省微生物菌种保藏中心(保藏号为GDMCC NO：61628，保藏地址：广东省广州市越秀区先烈中路100号)。
18S rRNA的核苷酸序列(SEQ ID NO：1)：
GAAGGGCGCTTACTGCGACCCTTTTCTTTCTACACATGTGTTTTTCTTTTTTTTG AAAACTTTGCTTTGGTGGGCCCACGGCCTGCCAGAGATTAAACTCAACCAAATTTTTTA TTAATTGTCAACTTGATTAACTAATAGTCAAAACTTTCAACAACGGATCTCTTGGTTCT CGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATATGAATTGCAGATATTCGTGA ATCATCGAATCTTTGAACGCACATTGCGCCCTTTGGTATTCCAAAGGGCATGCCTGTTTGAGCGTCATTTCTCCCTCAAACCTTCGGGTTTGGTGTTGAGCGATACGCTGGGTTTGCT TGAAAGAAAGGCGGAGTATAAACTAATGGATAGGTTTTTTTCTTCCACTCATTGGTACA AACTCCAAACATTCTTCCAAATTCGACCTCAAATCAGGTAGGACTACCCGCTGAACTTA AGCATATCAAAAGGGCGGAGGAA。
实施例2Candida metapsilosis M2006B对野生型小鼠炎性肠病的缓解作用
6-8周雄性C57BL/6J小鼠，适应性饲养1周后，随机分为四组 (对照组、对照组+Candida metapsilosis M2006B定植、炎性肠病造模组、造模组+Candida metapsilosisM2006B定植)，每组6只。
第1-14天，将Candida metapsilosis M2006B菌液(5×109CFU/Kg) 通过灌胃给予小鼠(对照组+M2006B、造模+M2006B)；对照组及炎性肠病造模组分别灌胃给予相同体积的无菌生理盐水。
第15-23天，四组小鼠均给予含有2.5％葡聚糖硫酸钠的饮用水，造炎性肠病模型。实验过程中，记录小鼠体重、活动指数、腹泻、便血以及直肠出血情况，结果如图2所示，其中A为小鼠体重变化；B 为小鼠腹泻评分；C为小鼠直肠出血或血便情况。由图2可知，与炎性肠病造组小鼠比较，M2006B定植小鼠表现出了较微弱的炎性肠病状态。
第23天，取各组小鼠进行结肠内镜检查，结果如图3A所示，与炎性肠病造模组小鼠相比，M2006B定植小鼠的结肠内壁光滑，溃疡病灶减少，出血症状改善。取肠组织进行HE染色，结果如图3B所示，与炎性肠病造模组小鼠相比，M2006B定植小鼠的结肠组织病理损伤明显减轻，肠绒毛结构恢复，炎症细胞浸润减少。将4组小鼠处死后，截取结肠，置于坐标纸上拍照，统计结肠长度，如图4A和4B所示， M2006B定植小鼠的结肠长度较为正常，显著长于炎性肠病造模组小鼠。取新鲜结肠组织，提取肠组织蛋白，通过western blot方法检测紧密连接蛋白Occludin和Claudin-2的表达，结果如图4C所示， M2006B定植改善DSS造模诱导的紧密连接蛋白表达下降，说明M2006B 定植组小鼠的结肠组织完整性较好。另取新鲜结肠组织，置于-80℃保存，经组织研磨仪提取RNA，反转录后，使用RT-PCR技术测定IL-1β(图5A)、IL-6(图5B)、TNF-α(图5C)、INF-γ(图5D)等炎症因子的表达情况，以评价小鼠肠道炎症反应情况。由图5可知，结果显示M2006B定植菌后，小鼠结肠组织的炎症水平显著下降，Candida metapsilosis M2006B对野生型炎性肠病小鼠结肠炎症反应具有改善作用。
实施例3Candida metapsilosis M2006B对无菌型小鼠炎性肠病的缓解作用
6-8周无菌雄性C57BL/6J小鼠产自于赛业生物技术有限公司，适应性饲养1周后，随机分为两组(炎性肠病造模组、造模组+Candida metapsilosis M2006B定植)，每组6只。
第1-14天，将Candida metapsilosis M2006B菌液(5×109 CFU/Kg)通过灌胃给予小鼠(造模+M2006B组)；炎性肠病造模组灌胃给予相同体积的无菌生理盐水。
第15-23天，两组小鼠均给予含有2.5％葡聚糖硫酸钠的饮用水，造炎性肠病模型。实验过程中，记录小鼠体重、活动指数、腹泻评分、便血以及直肠出血或血便情况。结果如图6所示，其中A为小鼠体重变化；B为小鼠腹泻评分；C为小鼠直肠出血或血便情况。由图6可知，与炎性肠病造组小鼠比较，M2006B定植小鼠表现出了较微弱的炎性肠病状态。
第23天，将两组小鼠处死后，截取结肠，置于坐标纸上拍照，统计结肠长度，结果表明M2006B定植小鼠的结肠长度较为正常，显著长于炎性肠病造模组小鼠(图7，A为照片，B为数据统计)，Candida metapsilosis M2006B对无菌型炎性肠病小鼠结肠组织长度的改善作用。取新鲜结肠组织，置于多聚甲醛固定液中，制石蜡切片，通过 HE染色，于显微镜下观察结肠组织炎症情况。HE染色切片如图8所示，与造模组(图8A)相比，M2006B定植组小鼠(图8B)的结肠组织完整性较好，且无明显的炎症浸润，Candida metapsilosis M2006B 对无菌型炎性肠病小鼠结肠病变的缓解作用。另取新鲜结肠组织，置于-80℃保存，取部分组织经组织研磨仪提取RNA，反转录后，使用 RT-PCR技术测定IL-1β(图9A)、IL-6(图9B)、TNF-α(图9C)、 INF-γ(图9D)等炎症因子的mRNA表达情况，以评价小鼠肠道炎症反应情况。结果显示M2006B定植菌后，小鼠结肠组织的炎症水平显著下降(图9)，Candida metapsilosisM2006B对无菌型炎性肠病小鼠结肠炎症反应具有改善作用。
实施例4Candida metapsilosis M2006B的安全性评价
6-8周雄性C57BL/6J小鼠，适应性饲养1周后，随机分为四组 (对照组、Candidametapsilosis M2006B定植组)，每组6只。
Candida metapsilosis M2006B定植组小鼠每天灌胃菌液(5× 109CFU/Kg)；对照组小鼠灌胃相同体积的无菌生理盐水。每天监测体重、精神状态和粪便状态，60天后，处死小鼠，取全血检测血常规。结果表明，菌定植期间，两组小鼠的体重、精神状态和粪便状态无明显差异。定植60天后，两组小鼠的血常规各项指标，包括红细胞、白细胞、血小板、血红蛋白计数等，均无显著差异(表1)。说明Candida metapsilosis M2006B长期定植没有毒性。
表1.Candida metapsilosis M2006B对小鼠血常规的影响
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
序列表
<110> 大连医科大学
<120> 一种肠道真菌Candida metapsilosis M2006B及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 491
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gaagggcgct tactgcgacc cttttctttc tacacatgtg tttttctttt ttttgaaaac 60
tttgctttgg tgggcccacg gcctgccaga gattaaactc aaccaaattt tttattaatt 120
gtcaacttga ttaactaata gtcaaaactt tcaacaacgg atctcttggt tctcgcatcg 180
atgaagaacg cagcgaaatg cgataagtaa tatgaattgc agatattcgt gaatcatcga 240
atctttgaac gcacattgcg ccctttggta ttccaaaggg catgcctgtt tgagcgtcat 300
ttctccctca aaccttcggg tttggtgttg agcgatacgc tgggtttgct tgaaagaaag 360
gcggagtata aactaatgga taggtttttt tcttccactc attggtacaa actccaaaca 420
ttcttccaaa ttcgacctca aatcaggtag gactacccgc tgaacttaag catatcaaaa 480
gggcggagga a 491
Claims (4)
1.一种肠道真菌Candida metapsilosis M2006B，其特征在于，所述肠道真菌Candidametapsilosis M2006B的菌种保藏号为GDMCC 61628。
2.一种如权利要求1所述的真菌Candida metapsilosis M2006B在制备预防和/或治疗溃疡性结肠炎或克罗恩病的药物中的应用。
3.根据权利要求2所述的应用，其特征在于，所述药物中还包括药学上可接受的载体。
4.根据权利要求2-3任一项所述的应用，其特征在于，所述真菌Candida metapsilosisM2006B的有效剂量为5×109CFU/Kg。
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