CN1183916C - 乳酸细菌在制备用于治疗和预防腹膜炎的载体中的用途 - Google Patents

乳酸细菌在制备用于治疗和预防腹膜炎的载体中的用途 Download PDF

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CN1183916C
CN1183916C CNB018071198A CN01807119A CN1183916C CN 1183916 C CN1183916 C CN 1183916C CN B018071198 A CNB018071198 A CN B018071198A CN 01807119 A CN01807119 A CN 01807119A CN 1183916 C CN1183916 C CN 1183916C
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E·施夫林
C·古尔纳
G·索里尔诺
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Abstract

本发明涉及应用能够与肠道粘膜粘附并主要在其上定植的乳酸细菌治疗和预防与腹膜炎相关的疾病。特别是，本发明涉及应用这样的乳酸细菌治疗和预防由于肝硬化导致的腹膜炎。

Description

乳酸细菌在制备用于治疗和预防腹膜炎的载体中的用途
本发明涉及应用能够与肠道粘膜粘附并主要在其上定植的乳酸细菌治疗与腹膜炎相关的疾病。特别是，本发明涉及应用这样的乳酸细菌治疗由于肝硬化导致的腹膜炎。
腹膜炎是腹膜的炎症，通常由于严重的局部感染导致，所述局部感染通常由胃肠道炎症和感染、胃肠道穿孔和创伤引起，包括手术和腹膜透析。在这种情况下，致病性和潜在致病性微生物，偶尔有细胞残留进入腹膜，引起宿主免疫应答，而这种免疫应答通常无法对付病原体的侵袭。
上述情况在肝硬化患者中更是如此。肝硬化患者通常有肠道细菌的过度生长和免疫应答的减弱，这似乎至少部分是由于腹水中调理素活性的减低。因此晚期肝硬化通常伴有自发性细菌性腹膜炎(SBP)，这种腹膜炎通常涉及正常情况下存在于肠道的革兰氏阴性肠道病原体。因此在腹水中发生SBP被认为是由于肠道细菌从肠道转位到腹膜导致的。
腹膜炎的主要危害是细菌播散，即病原体通过血液和淋巴系统的扩散，引起多数组织感染，导致受累个体发生威胁生命的状况。一旦细菌进入腹腔，播散就相当迅速。在狗腹腔内孵育细菌6分钟，胸部淋巴培养即呈阳性，而孵育12分钟则可导致血液细菌水平的上升。
尽管在腹膜炎的诊断和治疗方面已经有了一些进步，但是仍有大约三分之一合并这种感染的住院患者最终死于消化道出血、肝功能衰竭或肝肾综合征。
目前，预防和/或治疗腹膜炎典型的医学手段包括抗生素治疗，特别是在手术前。但是这种方法的缺点是可能引起导致腹膜炎的耐药细菌的产生。而且，由于腹膜炎可能由革兰氏阳性和革兰氏阴性两种细菌导致，应用抗生素可能不会在所有病例中发挥足够的功效。
此外，抗生素治疗是非特异性的，还可导致多种非致病性微生物的损伤，而这些非致病性微生物通常可以通过细菌拮抗作用预防细菌性疾病，特别是胃肠道疾病。因此，应用广谱抗生素一段较长时间，可以抑制绝大多数肠道内生存细菌的生长，而致病性微生物中的抗生素耐药菌株则可自由繁殖。导致的结果是，抗生素有时可能会使腹膜炎恶化，而非预防其发生。
WO 97/00081披露了另一种腹膜炎的治疗方法。该文件提示可以应用CD14拮抗剂，CD14是已知能够与细菌脂多糖发生相互作用的表面抗原。已知细菌脂多糖能够通过与免疫系统细胞的表面受体结合，刺激免疫系统，开始产生并分泌细胞因子和其他介质，来刺激免疫系统。而业已发现，这些细胞因子和介质可以支持细菌的生长和侵袭。因此提议的治疗主旨在于干扰产生细胞因子/介质的早期免疫应答。据信通过阻断CD14的激活，基本上防止细胞因子的产生，还可以抑制细菌的生长。但这种方法的缺点在于宿主的免疫应答功能也受到大大损伤，宿主自身的防御机制进一步恶化。
因此，有需要寻求一种有效的方法，预防和/或治疗局部感染，如与腹膜炎相关的疾病。因此本发明的一个目标就是提供这样一种方法。
在导向本发明的大量实验中，研究人员探讨了这样一个理论，即个体肠道中的乳酸细菌对腹膜中的免疫系统细胞特别是临近肠道的免疫系统细胞是否具有正面影响。业已发现，应用特定的乳酸细菌可以分别明显降低腹膜炎动物腹水和腹膜中的细菌数量，提示乳酸细菌，特别是益生性乳酸细菌，能够有效减少肠道细菌的过度生长和病原体的转位。
因此，可以通过应用乳酸细菌解决上述问题，所述乳酸细菌能够粘附于肠道粘膜，并主要在其上定植，制备成可吸收载体，用于预防和/或治疗与腹膜炎相关的疾病。
腹膜炎可以是自发性细菌性腹膜炎(SBP)，如在腹膜中突然出现潜在病原体导致的腹膜炎，这种情况通常在肝功能损伤的情况下发生，如在肝硬化期间。而且，腹膜炎还可能是由病毒诱导的，或者在透析或手术后发生。
根据一个优选实施方案，乳酸细菌是益生菌，优选乳杆菌和双岐杆菌属，更优选约氏乳杆菌(Lactobacillus johnsonii)CNCM I-1225、类干酪乳杆菌(Lactobacillus paracasei)CNCM I-2116或双岐杆菌(Biffdobacterium)CNCM I-2168。
根据本发明，所用乳酸细菌被整合到载体中，所述载体可以是食品或药品，如乳类，酸奶，凝乳，奶酪，发酵乳，乳基发酵制品，冰淇淋，发酵谷物基产品，奶粉，婴儿配方以及片剂，细菌悬液，干的口服添加剂，湿的口服添加剂，干的管饲物和湿的管饲物等。
载体中还可以包括已知对损伤的肠道状况有益的其他化合物，如抗氧化剂，如维生素C，维生素E，硒或锌。根据各自的治疗和/或预防性治疗的需要，本领域技术人员可以选择适宜的医学形式和/或添加剂，帮助改善个体的健康状况。
不受任何理论的限制，据信微生物的活性主要取决于两个因素：
已知益生菌对宿主的免疫系统具有有益作用。在目前情况下，乳酸细菌似乎能够刺激腹膜免疫系统，特别是位于个体肠道周围的非特异性免疫防御系统，抵御微生物通过肠壁对腹膜的侵袭。该免疫屏障在罹患肝硬化的患者中通常减弱。
另一方面，据信定植于肠道壁的乳酸细菌可以物理性防止或者至少降低潜在病原体通过肠道对腹膜的侵袭。通过与肠道的粘附，细菌可以与肠道表面的病原体肠道粘膜遮蔽受体建立紧密的联系。
应该指出，微生物活性呈剂量依赖性，即整合到可吸收食品或药物组合物中的微生物越多，保护性和/或治愈活性越高。因此，本发明所用食品或药物组合物可以包含的乳酸细菌数量大约为105cfu/g载体-1012cfu/g载体。本发明所用的缩略语“cfu”是指“集落形成单位”，其定义为通过琼脂平板上微生物计数获得的细菌细胞数量。
由于应用的微生物对于人类和动物来说是无害的，而且最终是从自然环境中分离的，即从婴儿粪便中分离，因此可以整合大量微生物，使微生物可以定植于个体肠道的绝大部分。
现在将以实施例的形式阐述本发明，但实施例不应被认为是对实施方案的限制。
实施例1
治疗动物
研究共包括31只雄性Sprague-Dawley大鼠，其中19只通过每周强制性灌胃诱发肝硬化，灌胃物为四氯化碳和含苯巴比妥的水(1.5mmol/l)随意。饲料为标准B&K(Barcelona，Spain)，大鼠可以随意取食。在整个研究期间，对照组的12只大鼠接受标准饲料(上述)和含苯巴比妥的水，而实验组其他大鼠(19)接受的标准饲料中含有大约1010cfu/天的乳酸细菌CNCM I-1225(约氏乳杆菌，La1)或水。
所有对照组大鼠在开始饮用含苯巴比妥的水18周后处死。12只对照大鼠中，有6只接受A处理(水)，6只接受B处理(水加乳杆菌)。处理通过强制手段实施。在分别接受处理或无处理后10天，麻醉进行剖腹手术，收集盲肠粪便、回肠粪便、回肠壁、肠系膜淋巴结、腹水和胸水样本进行微生物学研究。从腔静脉收集血液，确定是否存在内毒素血症。此外，还收集回肠壁、盲肠壁、脾脏和肝脏样本进行组织学研究，门脉压通过门静脉压力和脾脏/大鼠重量比率进行评价。
对于实验组，当大鼠产生腹水后(大约在开始诱导肝硬化后20周)，麻醉进行腹腔穿刺，核实腹水的存在，然后将这些大鼠随机分为两组：一组(n＝8)接受A处理(水)，另一组(n＝11)接受B处理(加乳酸细菌)。处理通过强制手段实施。研究结束时(处理后10天)，病程在对照组大鼠中相同。
在所有对照和肝硬化大鼠中，在开始处理前24小时和处理结束时，通过测定尿中乳果糖和甘露醇，进行了肠道渗透性研究。而且，在肝硬化诱导期或对照期，对所有对照组和肝硬化大鼠每4周进行一次肠道渗透性研究。
实施例2
腹水、细菌转位和细菌性腹膜炎的出现
在剖腹手术中，7/8应用水处理(处理A)的肝硬化大鼠和7/11处理B的肝硬化大鼠具有可检测的腹水。肠系膜淋巴结培养在所有22只对照大鼠中均为阴性。在所有接受B处理的肝硬化大鼠中，肠系膜淋巴结、腹水和胸水培养也均呈阴性。而接受水处理的肝硬化大鼠，5/8大鼠显示有细菌转位(培养呈阳性)，转移到肠系膜淋巴结或腹水或胸水(在接受A和B处理的组间，p＜0.01)：2只转位到胸水，2只转位到肠系膜淋巴结，1只转位到腹水和肠系膜淋巴结。分离出的细菌为：2例大肠杆菌，2例大肠杆菌+肠球菌，1例大肠杆菌+链球菌。
结果见下表I：
表I
  处理A(水)n＝8 处理(+乳酸细菌)n＝11
腹水     7/8     7/11
肠系膜淋巴结     3     0
腹水     2     0
胸水     2     0
总计     7/8     0/11
从上面的结果可以明显看出，接受约氏乳杆菌CNCM 1225的肝硬化大鼠相较于接受水的肝硬化大鼠，细菌转位明显受到抑制。
实施例3
肠道微生物学研究
在该研究中，测试了多种微生物，用于上述实施例1和2，处理对照和肝硬化大鼠。
    对照大鼠             肝硬化大鼠
Log 10CFU/g     处理Bn＝6     水n＝8     处理Bn＝11
盲肠类细菌     5.1±0.7     7.9±0.5     7.4±0.4
盲肠双叉乳杆菌     3.72±0.29     nd     ＜3.3
盲肠乳杆菌     7.3±0.1     nd     8.0±0.2
盲肠肠球菌     3.8±0.1     5.8±0.2     4.6±0.3#
盲肠肠杆菌     4.3±0.2     4.7±0.3     4.8±0.5
回肠类细菌     4.2±0.6     5.7±0.9     4.7±0.3
回肠双叉乳杆菌     ＜3.3     ＜3.3
回肠乳杆菌     7.3±0.1     6.4±0.4     7.8±0.3
回肠肠球菌     3.5±0.1     6.1±0.5     3.7±0.2##
回肠肠杆菌     3.4±0.0     5.3±0.6     3.7±0.6#
回肠壁类细菌     3.3±0.0     3.8±0.5     3.8±0.2
回肠壁双叉乳杆菌     ＜3.3     ＜3.3
回肠壁乳杆菌     5.6±0.2     6.4±0.3
回肠壁肠球菌     ＜3.3     4.4±0.6     ＜3.3##
回肠壁肠杆菌     3.4±0.1     3.5±0.2     ＜3.3
与水处理的肝硬化大鼠相比，*P＜0.01，**p＜0.001和***p＜0.03(Mann-Whitney检验)。
与水处理的肝硬化大鼠相比，#p＜0.05和##p＜0.01。
nd＝未测
上述数据还证实肠道微生物平衡确实存在损伤，即与对照大鼠相比，肝硬化大鼠中潜在致病性细菌存在过度生长。与水处理组大鼠相比，处理B降低了肠细菌和肠球菌的数量，增加了乳杆菌的数量。因此，处理B可能纠正肝硬化大鼠中潜在致病性细菌的过度生长。

Claims (9)

1.能够与肠道粘膜粘附并主要在其上定植的乳酸细菌在制备可吸收载体中的用途，所述载体用来预防和/或治疗与腹膜炎相关的疾病。
2.权利要求1的用途，其中所述乳酸细菌是益生菌。
3.权利要求1或2的用途，其中所述细菌选自乳杆菌或双岐杆菌。
4.权利要求3的用途，其中所述细菌是约氏乳杆菌CNCMI-1225，类干酪乳杆菌CNCMI-2116或双岐杆菌CNCMI-2168。
5.权利要求1或2的用途，其中所述腹膜炎是自发性细菌性腹膜炎，病毒诱导的腹膜炎或透析诱导的腹膜炎。
6.权利要求1或2的用途，其中所述腹膜炎与肝硬化相关。
7.权利要求1或2的用途，其中载体中所含的乳酸细菌量为105cfu/g载体-1012cfu/g载体。
8.权利要求7的用途，其中含有乳酸细菌的载体是食品或药物组合物。
9.权利要求7的用途，其中载体是食品组合物，选自乳类，酸奶，凝乳，奶酪，发酵乳，乳基发酵制品，冰淇淋，发酵谷物基产品，奶粉，婴儿配方，片剂，细菌悬液，干的口服添加剂，湿的口服添加剂，干的管饲物和湿的管饲物。
CNB018071198A 2000-03-24 2001-03-22 乳酸细菌在制备用于治疗和预防腹膜炎的载体中的用途 Expired - Fee Related CN1183916C (zh)

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