CN114246886B - 规则粪球菌在制备预防和治疗急性结肠炎的制剂中的应用 - Google Patents
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Abstract
本发明公开了规则粪球菌在制备预防和治疗急性结肠炎的制剂中的应用，所述规则粪球菌菌株购于美国模式培养物集存库(American Type CultureCollection,ATCC)，保藏号：ATCC27759。实验结果表明：在DSS诱导的结肠炎小鼠模型中，规则粪球菌ATCC27759能够显著改善小鼠疾病活跃指数、抑制脾肿大、降低肠道炎症因子的表达；规则粪球菌ATCC27759能够显著增加结肠炎小鼠肠道粘液分泌和紧密连接蛋白的表达而改善肠道屏障功能，并缓解急性结肠炎。该规则粪球菌ATCC27759有潜力开发为预防结肠炎、保护肠道屏障、调理肠道健康的益生菌制剂或功能性食品。
Description
技术领域
本发明涉及微生物领域，具体涉及规则粪球菌在制备预防和治疗急性结肠炎的制剂中的应用。
背景技术
结肠炎是一种慢性非特异性炎症性疾病，发病部位涉及回肠、结肠和直肠，临床表现为腹痛、呕吐、腹泻、血便或体重减轻等。由于病因复杂、反复发作、迁延不愈的特点，结肠炎已成为严重影响人类生活健康的一种疾病，加之近年来我国患病人数增加显著，且发病人群呈年轻化的态势，预计到2025年，全国结肠炎患者将达到150万人。而肠道菌群对于保护肠道功能完整，调理肠道健康，维持宿主稳态至关重要。近年来，肠道健康受到重视，肠道微生物研究的热潮也推动了益生菌研究的发展，肠道内的益生菌如双歧杆菌、乳酸杆菌等通过合成多种营养物质促进肠道稳态，抑制致病菌的生长。因此，开发针对性的能够改善或缓解结肠炎的功能益生菌，有助于推动益生菌的研发前景和产业化进程。
粪球菌属是存在于人体肠道中的梭菌目、毛螺菌科的革兰氏阳性厌氧菌，该菌属可以通过产生短链脂肪酸促进人体健康。规则粪球菌是粪球菌属的模式菌种，是一种专性厌氧菌，形态呈球状，通常成对出现。研究显示，肠道中的规则粪球菌的丰度减少与多种疾病如Ⅱ型糖尿病、乳腺癌和克罗恩病密切相关，服用抗生素环丙沙星也会引起肠道菌群中规则粪球菌的降低。这些发现显示了规则粪球菌有潜力作为评估人类胃肠道健康的微生物生物标志物。但关于规则粪球菌在维护肠道健康、改善急性结肠炎方面尚未报道。
发明内容
基于以上背景，本发明的目的在于提供规则粪球菌在制备预防和/或治疗急性结肠炎的益生菌制剂或功能性食品中的应用。
本发明提供规则粪球菌在制备预防和/或治疗急性结肠炎的益生菌制剂或功能性食品中的应用。所述的规则粪球菌(Coprococcus.eutactus)菌株购于美国模式培养物集存库(American Type CultureCollection,ATCC)，保藏号：ATCC27759，产品批号：70036335。
所述规则粪球菌ATCC27759在含有5％无菌脱纤维绵羊血的胰蛋白胨大豆固体培养基上菌落形态突起，边缘光滑、乳白色、溶血或微溶血。
所述规则粪球菌ATCC27759在预防和治疗急性结肠炎中的应用，具体是改善结肠炎的疾病状态、抑制炎症因子的表达和保护结肠屏障。所述的规则粪球菌ATCC27759在益生菌制剂中添加的活菌数应高于1×109CFU/mL，所述的规则粪球菌ATCC27759在益生菌功能性食品中添加的活菌数应高于1×109CFU/g。
本发明的有益效果：通过DSS诱导构建结肠炎小鼠模型，规则粪球菌ATCC27759在含有5％无菌脱纤维绵羊血的胰蛋白胨大豆液体培养基中培养，使菌落数达到109CFU/mL，灌胃小鼠108CFU，实验结果表明：在DSS诱导的结肠炎小鼠模型中，规则粪球菌ATCC27759能够显著改善小鼠疾病活跃指数、抑制脾肿大、降低肠道炎症因子的表达；规则粪球菌ATCC27759能够显著增加结肠炎小鼠肠道粘液分泌和紧密连接蛋白的表达而改善肠道屏障功能，并缓解急性结肠炎。该规则粪球菌ATCC27759有潜力开发为预防结肠炎、保护肠道屏障、调理肠道健康的益生菌制剂或功能性食品。
附图说明
图1规则粪球菌ATCC27759菌落形态
图2规则粪球菌ATCC27759灌胃后结肠炎小鼠疾病活跃指数变化图
图3规则粪球菌ATCC27759灌胃后DSS诱导的结肠炎小鼠脾脏重量统计图
图4规则粪球菌ATCC27759灌胃后小鼠肠道炎症因子水平统计图
图5阿利新蓝-过碘酸西弗染色检测规则粪球菌ATCC27759灌胃后小鼠肠道粘液展示图
图6 qPCR检测规则粪球菌ATCC27759处理后肠道紧密连接蛋白表达的统计柱状图；
图1～6中，“*”、“**”表示显著性差异水平，数据以Mean±SD形式展现。
具体实施方式
实施例1：规则粪球菌的复苏和扩大培养
1、规则粪球菌的复苏
(1)配置胰蛋白胨大豆液体培养基，在121℃高压蒸汽灭菌后，待培养基冷却至47℃时，加入5％的无菌脱纤维绵羊血，将含有5％无菌脱纤维绵羊血的胰蛋白胨大豆液体培养基置于厌氧培养箱中除去氧气并过夜静置；
(2)安瓿管装的规则粪球菌ATCC27759干粉购自美国模式培养物集存库，产品批号：70036335。在厌氧条件下，向安瓿管中滴加0.5mL灭菌后的含5％无菌脱纤维绵羊血的胰蛋白胨大豆液体培养基将冻干菌粉全部溶解，溶解后的菌悬液转移至盛有4mL液体培养基的试管中混匀，并吸取1滴菌悬液转接至固体培养基上，在37℃、严格厌氧(90％N2、5％CO2和5％H2)的条件下倒置培养12h后观察，发现固体培养基上看到菌落长出，说明菌株活化成功(如图1所示)。
2、规则粪球菌的扩大培养
(1)取步骤1中的菌落平板，选取单一菌落且生长状态好的菌斑，使用无菌接种环挑取平板上的单一菌斑并接种于步骤1(1)中的含有5％无菌脱纤维绵羊血的胰蛋白胨大豆液体培养基中，在严格厌氧条件下，置于37℃、200rpm/min的摇床中；
(2)摇菌16h后,在厌氧培养箱中取(1)中液体培养基100μL溶于900μL无菌PBS中，标记编号为1，浓度为10-1，稀释倍数为101倍；在编号1中吸取100μL溶于900μL无菌PBS中，标记编号为2，浓度为10-2，稀释倍数为102倍；重复梯度稀释直至浓度为10-8,取10-6、10-7、10-8、浓度涂布于含有5％无菌脱纤维绵羊血的胰蛋白胨大豆琼脂平板上，每个浓度三个重复，在厌氧培养箱中倒置培养12h；
(3)平板CFU计数：选取(2)中平板菌落生长均匀且单菌落数在10-300之间的平板进行菌落计数，液体培养基中活菌数量为菌落数乘以稀释倍数；最终菌浓度为1x109CFU/mL；
(4)菌液分装与保存：将(3)中计数后的菌悬液吸取1mL置于新的无菌EP管中，4℃8000rpm/min离心10min,离心结束后弃掉上清并使用1mL无菌PBS充分重悬沉淀，待混匀后取吸取100μL到新的无菌EP管中，并于-80℃冰箱保存，保存的菌悬液最终浓度为1x109CFU/mL。分装的菌悬液在-80℃冰箱保存的时间在半年以内。
实施例2：规则粪球菌改善DSS诱导的结肠炎小鼠疾病活跃指数升高和脾肿大
1、动物来源：雄性SPF级C57BL/6小鼠，6-8周龄；
2、饲养条件和饲料：温度为23±2℃，相对湿度为50-55％，室内灯光模拟昼夜，12小时明暗循环。正常对照组为正常饮水，除正常对照组外均DSS饮水:先正常饮水一周，然后饮用含3％DSS的水一周，持续2周。ATCC27759处理组采用灌胃方法，给小鼠每日灌胃的菌液中ATCC27759活菌数为1×109CFU/mL，每只小鼠灌胃100μL，对照和模型组灌胃同等体积无菌PBS，直到实验结束。
3、实验动物分组(见表1)：
表1实验分组
4、实验进行过程中每天对小鼠进行称重，粪便观察和收集，并记录数据，统计小鼠疾病活跃指数并绘制折线图，具体方法为：每天观察并收集小鼠粪便，并对粪便是否成形以及是否有便血进行打分(打分标准见表2)。疾病活跃指数为体重下降、粪便黏稠度和便血的评分之和。
表2疾病活跃指数评分表
结果表明，与模型组相比，规则粪球菌ATCC27759处理后的结肠炎小鼠疾病活跃指数明显降低(如图2所示)。
5、小鼠脾脏的称重，麻醉处死小鼠后，解剖小鼠并迅速取出脾脏组织，去除其他组织和脂肪残留后，在干净无菌的条件下称量脾脏重量并记录。然后迅速将整个脾脏组织投入液氮中速冻并放于-80℃长期保存。结果表明，与模型组相比，规则粪球菌ATCC27759处理能明显抑制结肠炎小鼠脾脏的肿大。(如图3所示)。
实施例3：规则粪球菌下调DSS诱导的结肠炎小鼠结肠炎症因子的表达
小鼠肠组织总RNA的提取：麻醉处死小鼠后，切取适量结肠组织10mm左右，将切好的组织用PBS清洗三遍，浸泡入1mL的Trizol中，匀浆后分装于进口EP管中(不立即提RNA的保存于-80℃)；加入200μL氯仿，剧烈振荡15s，静置5min，于4℃ 13,000rpm下离心15min，至分层(无色水相、酚-氯仿相、浅红色相)吸取上清存放在灭菌的EP管中；加入等体积的异丙醇，上下颠倒离心管，充分混匀，在室温环境下，静置10min；于4℃ 13,000rpm下离心10min，见白色沉淀；小心倒掉上清，沿管壁缓慢加入1mL 75％的乙醇(可轻轻上下颠倒)；于4℃13,000rpm下离心15min，丢弃上清，并用枪头吸干净；吹干沉淀，直至变为透明；加入适量DEPC水，溶解沉淀，在57℃金属浴助溶10min；
RNA浓度检测：使用Nanodrop 2000测定RNA浓度。
RNA模板反转录：将上述方法提取的总RNA，用HiScriptⅡReverse Transcriptase反转录试剂盒，进行反转录，反应条件为:37℃ 15min,85℃ 5s，反应结束后，cDNA存放于-20℃长期保存。
实时定量PCR：设计并合成小鼠源的紧密连接蛋白的引物，使用ChamQ SYBR qPCRMaster Mix试剂盒进行实时定量PCR。
结果表明：与对照组相比，DSS结肠小鼠炎症因子TNF-α、IL-1β以及IL-6表达均显著升高，规则粪球菌ATCC27759处理可以明显降低DSS结肠炎小鼠肠道中炎症因子的表达水平(如图4所示)。
实施例4：规则粪球菌改善DSS诱导的结肠炎小鼠的黏膜屏障
小鼠结肠组织的阿利新蓝-过碘酸雪夫染色分析：麻醉处死小鼠后，将小鼠解剖，取其结肠部位并置于组织固定液后做成石蜡切片。依次将切片放入二甲苯Ⅰ20min-二甲苯Ⅱ20min-无水乙醇Ⅰ5min无水乙醇Ⅱ5min-75％酒精5min，自来水洗。使用AB-PAS染液试剂盒进行染色：(1)切片入AB-PAS染液C浸染8-10min，自来水洗至玻片流水呈无色；(2)切片进入AB-PAS染液B浸染10-15min，经纯水浸洗3次，每次约10s；(3)切片进入AB-PAS染液A(提前恢复至室温)中加盖避光浸染25-30min，流水冲洗5min。然后进行脱水封片：切片依次入无水乙醇I 5min-无水乙醇II 5min-无水乙醇Ⅲ5min-二甲苯Ⅰ5min-二甲苯Ⅱ5min透明，中性树胶封片。最后在显微镜下进行镜检和图像采集。
结果表明：模型组和对照组相比，结肠中黏液的分泌明显减少，说明DSS诱导的炎症引起小鼠肠道黏膜的破坏；而且规则粪球菌ATCC27759可以有效阻止肠道黏液分泌的减少(如图5所示规则粪球菌ATCC27759处理组蓝色黏液明显增加)。
实施例5：规则粪球菌改善DSS诱导的结肠炎小鼠肠道紧密连接蛋白的表达
按照实例3中的方法提取小鼠肠组织中的RNA，提取RNA并反转录成cDNA后，进行实时定量PCR，设计并合成小鼠源的紧密连接蛋白的引物，使用ChamQ SYBR qPCR Master Mix试剂盒进行实时定量PCR。
结果表明：与对照组相比，DSS诱导结肠炎小鼠紧密连接蛋白Claudin-1和Occludin表达均显著降低，规则粪球菌ATCC27759处理可以明显增加DSS诱导的结肠炎小鼠肠道中紧密连接蛋白的表达水平(如图6所示)。
Claims (2)
1.规则粪球菌在制备以其为唯一活性成分的预防和/或治疗急性结肠炎的益生菌制剂中的应用；所述的规则粪球菌保藏号为ATCC27759。
2.如权利要求1所述的应用，其特征在于，所述的规则粪球菌ATCC27759在益生菌制剂中添加的活菌数高于1×109 CFU/mL。
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