CN110585187A - 绿原酸在抑制铜绿假单胞菌胞内c-di-GMP低表达运动中的应用 - Google Patents
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Abstract
本发明公开了绿原酸在抑制铜绿假单胞菌胞内c‑di‑GMP低表达运动中的应用，并具体提供了抑制方法和绿原酸的浓度。本发明证明了绿原酸能够抑制铜绿假单胞菌胞内c‑di‑GMP表达，从而抑制铜绿假单胞菌的运动感。通过本发明能够抑制铜绿假单胞菌的运动，并且为寻找有效干预铜绿假单胞菌的药物作用靶点提供依据。
Description
技术领域
本发明涉及一种抑制菌类生物膜产生的方法，属于微生物技术领域。
背景技术
人类诸多感染性疾病的研究表明，致病菌在人体内存在的形式主要是以生物被膜的状态存在，目前研究表明生物被膜对于人体的致病力远远高于浮游菌，国内外研究表明抗生素对于生物被膜的抑制作用所需剂量为对浮游菌抑制作用所需剂量的30-2000倍。研究表明，慢阻肺合并肺内感染患者及肺囊性纤维化化患者肺内长期定植菌常见为铜绿假单胞菌，其中铜绿假单胞菌混合烟曲霉菌感染是最常见的混合感染类型，两者往往以混合生物被膜的形式存在于患者肺内，因而抗炎治疗效果欠佳。
中药及中药成分联合抗生素的应用近年来愈发受到医学界的重视，金银花主要抗真菌成分为绿原酸和异绿原酸，绿原酸可破坏铜绿假单胞菌及烟曲霉生物被膜。对于目前临床用药具有参考价值，临床治疗烟曲霉菌、铜绿假单胞菌混合感染的效果欠佳，常见抗真菌药物的肝肾损害较明显且副作用较多。易造成疾病迁延不愈增加死亡率。
铜绿假单胞菌为条件致病菌，是医院内感染的主要病原菌之一。患代谢性疾病、血液病和恶性肿瘤的患者，以及术后或某些治疗后的患者易感染本菌。
经常引起术后伤口感染，也可引起褥疮、脓肿、化脓性中耳炎等。本菌引起的感染病灶可导致血行散播，而发生菌血症和败血症。烧伤后感染了铜绿色假单胞菌可造成死亡。
本菌普遍存在，而在潮湿环境尤甚。铜绿假单胞菌是存在于人类中最常见的一种假单胞菌，它偶尔可在腋下和肛门生殖道周围的正常皮肤，但除非给服抗生素，在粪中甚为罕见。该菌通常伴随毒力较强的细菌存在于病灶中，但偶尔也可单独引起暴露于外部的组织感染。感染通常发生于医院内，洗涤槽、防腐溶液和贮尿容器中常可发现这种细菌。通过医护人员可将病菌传给病人，特别在灼伤和新生儿重症监护室。是重要的医院内病原菌。
在体内铜绿假单胞菌生物被膜感染过程中，其c-di-GMP信号通路是新兴且有前景的研究方向，
C-di-GMP目前被认为是细菌生活方式转变的主要调控因子,铜绿假单胞菌的表面附着、运动控制(泳动、集群运动和蹭行运动)以及细胞外基质成分的产生有关。每个过程都在一定程度上由c-di-GMP信号通路控制，它的运动性对于它本身对环境的探索和环境表面的接触到生物被膜的形成有着重要作用。
对临床铜绿假单胞菌感染的防治具有较大的指导意义。
发明内容
本发明的目的是提供一种体外绿原酸抑制铜绿假单胞菌胞内c-di-GMP低表达运动的方法，以弥补现有技术的不足。
为达到上述目的，本发明采取的具体技术方案为：
绿原酸在抑制铜绿假单胞菌胞内c-di-GMP低表达运动中的应用。
一种体外绿原酸抑制铜绿假单胞菌胞内c-di-GMP低表达运动的方法，该方法利用绿原酸进行抑制铜绿假单胞菌胞内c-di-GMP低表达运动的形成。
进一步的，上述绿原酸的浓度为1024-4096μg/mL。
进一步地，上述绿原酸的浓度为4096μg/mL。
本发明的优点和技术效果：
本发明证明了绿原酸能够抑制铜绿假单胞菌胞内c-di-GMP表达，从而抑制铜绿假单胞菌的运动感。
通过本发明能够抑制铜绿假单胞菌的运动，并且为寻找有效干预铜绿假单胞菌的药物作用靶点提供依据。
附图说明
图1为不同浓度绿原酸干预胞内c-di-GMP低表达铜绿假单胞菌集群运动下的数据结果图。
图2为不同浓度绿原酸干预胞内c-di-GMP低表达铜绿假单胞菌泳动下的数据结果图。
图3为不同浓度绿原酸干预胞内c-di-GMP低表达铜绿假单胞菌蹭行运动下的数据结果图。
图4为不同浓度绿原酸干预胞内c-di-GMP低表达铜绿假单胞菌集群运动下的观察结果图。
图5为不同浓度绿原酸干预胞内c-di-GMP低表达铜绿假单胞菌泳动下的观察结果图。
图6为不同浓度绿原酸干预胞内c-di-GMP低表达铜绿假单胞菌蹭行运动下的观察结果图。
具体实施方式
以下通过具体实施例并结合附图对本发明进一步解释和说明。
实施例1：干预试验
用浓度为4096μg/mL的绿源酸干预胞内c-di-GMP低表达铜绿假单胞菌，可以明显抑制其泳动(swimming)、集群运动(swarmming)、蹭行运动(twitching)。
由图1-图3可以看出，在4096μg/mL时，绿原酸对它的运动性有着很好的抑制作用，也就是说4096μg/mL是最优值，在这个浓度上均能对它的运动性有着很好的抑制效果。(Y是低表达菌株的简写)
实施例2：验证该方法的有效性
铜绿假单胞菌主要的运动方式有三种，分别是泳动(swimming)、集群运动(swarmming)、蹭行运动(twitching)，以下是绿原酸干预详细方案。
(1)泳动干预
用10ɡ/L胰蛋白胨、5ɡ/L氯化钠、0.3％(wt/vol)琼脂制成培养基，以上培养基中，干预组分别含有浓度为4096μg/mL,2048μg/mL，1024μg/mL绿原酸。用无菌牙签挑起菌落，接种于培养基中间层，置于恒温37℃培养箱，培养24小时后，观察以接种中心蔓延的云雾状区域。
如图2所示，从左往右的干预浓度为4096μg/mL到1024μg/mL，最右是空白组。在4096μg/mL时，绿原酸对它的运动性有着很好的抑制作用，几乎是完全抑制，也就是说4096μg/mL是最优值，在这个浓度上均能对它的运动性有着很好的抑制效果。(Y是低表达菌株的简写)。
(2)集群运动干预
用10ɡ/L胰蛋白胨、5ɡ/L氯化钠、0.5％(wt/vol)琼脂制成培养基，以上培养基中，干预组分别含有浓度为4096μg/mL,2048μg/mL，1024μg/mL绿原酸。将隔夜培养后的胞内c-di-GMP低表达铜绿假单胞菌菌株用LB肉汤稀释各菌株至OD600＝0.05，分别用移液枪吸取5μl菌液接种于上述培养基表面，置于恒温37℃培养箱，培养24小时后，观察以接种中心生长蔓延的区域。
如图1所示，从左往右的干预浓度为4096μg/mL到1024μg/mL，最右是空白组。在4096μg/mL时，绿原酸对它的运动性有着很好的抑制作用，几乎是完全抑制，也就是说4096μg/mL是最优值，在这个浓度上均能对它的运动性有着很好的抑制效果。(Y是低表达菌株的简写)
(3)蹭行运动干预
用10ɡ/L胰蛋白胨、5ɡ/L氯化钠、1％(wt/vol)琼脂、5ɡ/L葡萄糖制成培养基，以上培养基中，干预组分别含有浓度为4096μg/mL,2048μg/mL，1024μg/mL绿原酸。用无菌牙签挑起菌落，接种于培养基底部，置于恒温37℃培养箱，培养24小时后，并轻轻揭去培养皿内琼脂，0.9％生理盐水冲洗培养皿底部未黏附的细菌，1％结晶紫染色10min，无菌双蒸水冲洗，然后观察以细菌接种点为中心形成的区域。
如图3所示，从左往右的干预浓度为4096μg/mL到1024μg/mL，最右是空白组。在4096μg/mL时，绿原酸对它的运动性有着很好的抑制作用，几乎是完全抑制，也就是说4096μg/mL是最优值，在这个浓度上均能对它的运动性有着很好的抑制效果。(Y是低表达菌株的简写)
综上，绿原酸可以抑制胞内c-di-GMP低表达铜绿假单胞菌菌株的运动，其运动方式主要有三种，分别是泳动(swimming)、群集(swarming)\、蹭动(twiching)。.绿原酸均能抑制这三种运动。
Claims (4)
1.绿原酸在抑制铜绿假单胞菌胞内c-di-GMP低表达运动中的应用。
2.一种体外绿原酸抑制铜绿假单胞菌胞内c-di-GMP低表达运动的方法，其特征在于，该方法利用绿原酸进行抑制铜绿假单胞菌胞内c-di-GMP低表达运动的形成。
3.如权利要求2所述的方法，其特征在于，所述绿原酸的浓度为1024-4096μg/mL。
4.如权利要求3所述的方法，其特征在于，所述绿原酸的浓度为4096μg/mL。
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| 温红侠等: "绿原酸对铜绿假单胞菌粘附功能的抑制作用的研究", 《中华医学会第12次全国内科学术会议论文汇编》, pages 116 - 117 * |
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