CN115212237B - 普拉梭菌在制备治疗心肌梗死后的心室病理性重构和/或心力衰竭的药物中的应用 - Google Patents
普拉梭菌在制备治疗心肌梗死后的心室病理性重构和/或心力衰竭的药物中的应用 Download PDFInfo
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Abstract
本发明提供了普拉梭菌(Faecalibacteriumprausnitzii)在制备治疗心肌梗死后的心室病理性重构和/或心力衰竭的药物中的应用,属于生物医药技术领域,所述普拉梭菌在实验动物水平上能够改善由于心肌梗死导致的心室病理性重构和/或心力衰竭,恢复心肌梗死小鼠的心脏收缩功能,降低心肌梗死小鼠的心脏纤维化,抑制心肌梗死小鼠的病理性心肌肥厚,且灭活的普拉梭菌不具有改善的作用。
Description
技术领域
本发明属于生物医药技术领域,尤其涉及普拉梭菌在制备改善心肌梗死后的心室病理性重构和/或心力衰竭的药物中的应用。
背景技术
心脏是人和脊椎动物体内推动血液循环的器官,其主要功能是为血液流动提供动力,把血液运行至身体各个部分。心肌梗死(Myocardial Infarction)是缺血性心脏病/冠状动脉疾病的常见表现,是世界范围内的主要死亡原因。心肌梗死主要是由于动脉粥样硬化斑块破裂导致冠状动脉管腔内血栓形成,阻塞流向远端心肌的血液,从而导致心肌细胞的死亡以及梗死区域组织的坏死并由疤痕组织取代死亡的心肌细胞。心肌梗死会改变心室的形态及功能,引起心室病理性重构,如心肌层厚度减少、心室扩张、梗死远端区的心肌细胞肥大以及心功能的下降,进一步的会发展成为心力衰竭并导致死亡。心肌梗死后心室的病理性重构是一个复杂的过程,涉及信号分子转导、细胞外基质、神经激素调节等众多过程。尽管目前有药物能够应对这一过程,但心肌梗死后心室重构导致的心力衰竭仍然具有极高的死亡率。因此在心肌梗死后保持心脏的收缩功能并防止不良的心室重构的发生是预防心力衰竭的关键一步。因此除了常用的临床手段外,防治心肌梗死后心室病理性重构仍需要新的途径和方法。
哺乳动物肠道内有着数量巨大的微生物群,包括细菌、古细菌、病毒和单细胞真核生物,其大部分生存于肠道内,被称为肠道菌群。肠道菌群与哺乳动物宿主长期共存形成共生关系,能够形成肠道表面屏障抑制致病菌的生长繁殖,调节宿主免疫功能,协助宿主消化食物提供维生素、脂肪酸等营养物质并控制营养物质的吸收,也能够通过代谢产生具有生物活性的信号分子来保持宿主健康或是引起疾病。肠道菌群会受到疾病的影响。在炎症性肠病、Ⅰ型及Ⅱ型糖尿病、动脉粥样硬化、高血压和心力衰竭患者中,肠道菌群多样性低于健康个体或是菌群的构成发生了改变,因而肠道菌群的多样性和组成对于哺乳动物宿主的健康非常重要。
普拉梭菌(Faecalibacterium prausnitzii)是厚壁菌门(Firmicutes)、梭状芽孢杆菌纲(Clostridium)、瘤胃球菌科(Ruminococcaceae)的物种,普遍存在于哺乳动物肠道内,对氧极度敏感。作为人体的共生厌氧菌之一,是成人肠道菌群中丰度最高的菌种,约占总菌群数的5%,主要发酵产物丁酸盐是结肠上皮细胞主要的能量物质。此外,Faecalibacterium prausnitzii还能调控宿主基因表达、抵抗炎症和促进肠道健康,有作为益生菌的潜力。在多种肠道及代谢疾病中,粪便中的Faecalibacterium prausnitzii丰度会降低,因而也具有作为生物标志物的潜力。
目前对于普拉梭菌(Faecalibacterium prausnitzii)的功能研究主要集中在肠道及代谢疾病,尚没有其他方面应用的研究。
发明内容
有鉴于此,本发明的目的在于提供普拉梭菌在制备治疗心肌梗死后的心室病理性重构和/或心力衰竭的药物中的应用;所述普拉梭菌在实验动物水平上能够改善由于心肌梗死导致的心室病理性重构和/或心力衰竭,恢复心肌梗死小鼠的心脏收缩功能,降低心肌梗死小鼠的心脏纤维化,抑制心肌梗死小鼠的病理性心肌肥厚。
本发明提供了普拉梭菌(Faecalibacterium prausnitzii)在制备治疗心肌梗死后的心室病理性重构和/或心力衰竭的药物中的应用。
优选的,所述普拉梭菌为普拉梭菌VPI C13-51,所述普拉梭菌VPI C13-51的ATCC保藏号为ATCC 27768。
优选的,所述普拉梭菌用改良强化梭菌培养基于严格厌氧环境中培养获得。
优选的,所述药物的剂型为液体制剂或固体制剂。
优选的,当所述药物的剂型为液体制剂时,所述液体制剂的活菌浓度为109~1012CFU/ml。
优选的,所述液体制剂的活菌浓度为1010~1011CFU/ml。
优选的,所述药物通过将所述普拉梭菌菌体悬浮于磷酸盐缓冲液获得。
优选的,所述磷酸盐缓冲液还包括体积百分含量为10%的甘油。
优选的,所述药物为口服制剂。
本发明提供了普拉梭菌(Faecalibacterium prausnitzii)在制备治疗心肌梗死后的心室病理性重构和/或心力衰竭的药物中的应用,所述普拉梭菌在实验动物水平上能够改善由于心肌梗死导致的心室病理性重构和/或心力衰竭,恢复心肌梗死小鼠的心脏收缩功能,降低心肌梗死小鼠的心脏纤维化,抑制心肌梗死小鼠的病理性心肌肥厚。
本发明将体外培养的普拉梭菌作为外源性的菌种补充回宿主肠道内,所述的普拉梭菌在宿主肠道内发挥作用,改善心肌梗死后心室病理性重构和/或心力衰竭。
进一步的,本发明将普拉梭菌作为液体制剂通过口服的方式作用于心肌梗死后的小鼠个体,所述普拉梭菌能够改善心肌梗死后心室的病理性重构并部分恢复心脏功能,实现将普拉梭菌作为益生菌或药物用于治疗心肌梗后心室病理性重构的目的。
进一步的,本发明提供了将所述的普拉梭菌在体外培养后,将其菌悬液回补于心肌梗死小鼠。小鼠心脏超声结果表明,心肌梗死小鼠在回补普拉梭菌后其心脏收缩功能增强,而回补灭活后的普拉梭菌对心脏的收缩功能没有恢复作用。在心肌细胞面积方面的影响,心肌梗死小鼠其心肌细胞发生病理性肥大,回补普拉梭菌后心肌细胞的病理性肥大程度得到抑制,但回补灭活后的普拉梭菌没有抑制心肌细胞病理性肥大的作用。从损伤区域的纤维化程度来看,回补普拉梭菌,损伤区域的纤维化程度有所减少,而回补灭活的普拉梭菌纤维化程度没有减少。本发明回补的普拉梭菌是具有生物活性的活菌,其非活性或死亡状态不会改善心肌梗死后心室病理性重构。
附图说明
图1为超声心动图检测Faecalibacterium prausnitzii的回补对心肌梗死小鼠心脏收缩功能的影响,其中A表示心肌梗死小鼠回补PBS后超声心动图结果;B表示心肌梗死小鼠回补Faecalibacterium prausnitzii后超声心动图结果;C表示肌梗死小鼠回补灭活的Faecalibacterium prausnitzii后超声心动图结果;D为回补PBS、Faecalibacteriumprausnitzii或灭活Faecalibacterium prausnitzii后心肌梗死小鼠左心室射血分数的统计结果,结果表明Faecalibacterium prausnitzii的回补能够提高心肌梗死小鼠左心室射血分数,而灭活组不具有这一效应;E为回补PBS、Faecalibacterium prausnitzii或灭活Faecalibacterium prausnitzii后心肌梗死小鼠左心室缩短分数的统计结果,结果表明Faecalibacterium prausnitzii的回补能够提高心肌梗死小鼠左心室缩短分数,而灭活组不具有这一效应。
图2为WGA染色检测Faecalibacterium prausnitzii的回补对心肌梗死小鼠心肌肌纤维横截面积的影响;其中A表示心肌梗死小鼠回补PBS后WGA染色结果图;B表示心肌梗死小鼠回补Faecalibacterium prausnitzii后WGA染色结果图;C表示肌梗死小鼠回补灭活的Faecalibacterium prausnitzii后WGA染色结果图;D为回补PBS、Faecalibacteriumprausnitzii或灭活Faecalibacterium prausnitzii后心肌梗死小鼠心肌肌纤维横截面积的统计结果,结果表明Faecalibacterium prausnitzii的回补能够减小心肌梗死小鼠心肌肌纤维横截面积,减轻心肌梗死小鼠的心肌病理性肥厚,而灭活组不具有这一效应。
图3为HE染色检测Faecalibacterium prausnitzii的回补对心肌梗死小鼠心肌肌纤维横截面积的影响;其中,A表示心肌梗死小鼠回补PBS后HE染色结果图;B表示心肌梗死小鼠回补Faecalibacterium prausnitzii后HE染色结果图;C表示肌梗死小鼠回补灭活的Faecalibacterium prausnitzii后HE染色结果图;D为回补PBS、Faecalibacteriumprausnitzii或灭活Faecalibacterium prausnitzii后心肌梗死小鼠心肌肌纤维横截面积的统计结果,结果表明Faecalibacterium prausnitzii的回补能够减小心肌梗死小鼠心肌肌纤维横截面积,减轻心肌梗死小鼠的心肌病理性肥厚,而灭活组不具有这一效应。
图4为马松染色检测Faecalibacterium prausnitzii的回补对心肌梗死小鼠心脏纤维化程度的影响;其中A表示心肌梗死小鼠回补PBS后马松染色结果图;B表示心肌梗死小鼠回补Faecalibacterium prausnitzii后马松染色结果图;C表示肌梗死小鼠回补灭活的Faecalibacterium prausnitzii后马松染色结果图;D为回补PBS、Faecalibacteriumprausnitzii或灭活Faecalibacterium prausnitzii后心肌梗死小鼠心脏纤维化的统计结果,结果表明Faecalibacterium prausnitzii的回补能够减轻心肌梗死小鼠心脏纤维化,而灭活组不具有这一效应。
具体实施方式
本发明提供了普拉梭菌(Faecalibacterium prausnitzii)在制备治疗心肌梗死后的心室病理性重构和/或心力衰竭的药物中的应用。
在本发明中,所述普拉梭菌为普拉梭菌VPI C13-51,所述普拉梭菌VPI C13-51的ATCC保藏号为ATCC 27768。在本发明具体实施过程中,所述普拉梭菌VPI C13-51优选的购自美国模式培养物集存库ATCC。
在本发明中,所述普拉梭菌用改良强化梭菌培养基于严格厌氧环境中培养获得;所述改良强化梭菌培养基优选的购自北京酷来搏科技有限公司(Coolaber,DZSL0529),所述改良强化梭菌培养基的成分组成如表1所示。
表1改良强化梭菌培养基的成分组成
在本发明中,所述所述改良强化梭菌培养基配制完成后,优选的分装至厌氧容器中进行灭菌;所述厌氧容器优选为厌氧培养管,所述灭菌优选为高温高压灭菌,所述灭菌的具体参数优选为0.1Mpa,121℃,20分钟。本发明在所述改良强化梭菌培养基灭菌后接种所述普拉梭菌进行严格厌氧培养,所述严格厌氧优选为在严格的厌氧环境下进行悬浮培养。在本发明中,所述厌氧培养方法优选的如下:厌氧培养操作于厌氧培养箱(ThermoScientific 1029厌氧培养箱)中进行。培养箱外接氮气和氢气后,首先通过重复置换培养箱气体20次以排尽大部分氧气,之后在培养箱中加入钯催化剂及硅胶颗粒,在钯催化剂作用下氢气与培养箱内剩余的氧气反应生成水并被硅胶吸收,以达到培养箱内严格的厌氧环境。在厌氧培养箱中,将购于ATCC的普拉梭菌VPI C13-51菌种干粉按产品说明书进行复苏。在厌氧培养箱中,取复苏后普拉梭菌菌液,用无菌接种环蘸取菌液后于改良强化梭菌固体培养基上划线培养。培养48小时后,挑取固体培养基上的单菌落进行悬浮培养。具体的,在厌氧培养箱中,取35ml改良强化梭菌培于50ml无菌离心管中,将挑取的单菌落置于其中培养48小时。
在本发明中,所述药物的剂型优选为液体制剂或固体制剂。当所述药物的剂型为液体制剂时,所述液体制剂的活菌浓度优选为109~1012CFU/ml,更优选为109~1011CFU/ml,最优选为5×1010CFU/ml。在本发明中,所述药物通过将所述普拉梭菌菌体悬浮于磷酸盐缓冲液获得。在本发明中,所述磷酸盐缓冲液优选为1×磷酸盐缓冲液,所述磷酸盐缓冲液的pH值优选为7.3;所述磷酸盐缓冲液优选的还包括体积百分含量为10%的甘油。在本发明中,所述甘油的作用是在低温条件下保护菌体活性。在本发明具体实施过程中,采用所述改良强化梭菌培养基培养获得所述普拉梭菌后,进行固液分离、收集菌体;然后用所述磷酸盐缓冲液重悬菌体至上述限定的活菌浓度范围。在本发明中,所述固液分离的方法优选为离心,所述离心的转速优选为8000~10000rpm,更优选为8500~9500rpm,最优选为9000rpm;所述离心的时间优选为8~12分钟,更优选为10分钟。本发明在所述离心后,收集菌体,用所述磷酸盐缓冲液重悬菌体。在本发明中,优选的用所述磷酸盐缓冲液重选菌体1~3次,更优选为2次,然后再次离心收集菌体,用含甘油的磷酸盐缓冲液重悬至上述限定的活菌浓度范围获得液体制剂;所述液体制剂可以直接使用或置于低温保存;所述低温保存的温度优选为-80℃。
在本发明中,所述药物优选为口服制剂;通过口服的方式给药。在本发明中,当给药对象为小鼠时,所述药物的给药浓度优选为108~109CFU/100μl,更优选为5×108CFU/100μl;所述药物的给药剂量优选为100~300μl/只小鼠,更优选为200μl/只小鼠;所述药物的给药方式为灌胃;所述药物优选的1周给药3次,持续8周。在本发明中,当给药对象为人时,给药优选为1×107CFU/天,给药方式为口服。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
普拉梭菌Faecalibacterium prausnitzii的培养及保藏
普拉梭菌Faecalibacterium prausnitzii菌株VPI C13-51购自ATCC (ATCC27768)。培养基采用改良强化梭菌培养基(ATCC Medium:2107Modified ReinforcedClostridial)。具体的,培养基购于北京酷来搏科技有限公司(Coolaber,DZSL0529),所述改良强化梭菌培养基的成分组成如表1所示。
采用表1所述培养基,分装至厌氧管中并经高温高压(0.1Mpa,121℃,20分钟)灭菌后于严格的厌氧环境下悬浮培养Faecalibacterium prausnitzii。
培养结束前采用上述的平板培养基培养Faecalibacterium prausnitzii进行计数。取1.5ml无菌EP管,用无菌PBS按浓度梯度稀释原菌液,稀释倍数分别为:10,100,1×103,1×104,1×105,1×106,1×107,1×108。具体的:用微量移液器取l00μl己充分混匀的菌悬液,加入至含有900μl无菌PBS的1.5mlEP管中,充分混匀,此即为10倍稀释;混匀后,从10倍稀释的EP管中取l00μl菌悬液,加入至含有900μl无菌PBS的EP管中,充分混匀,此即为100倍稀释;此后的稀释即是从上一个稀释倍数的EP管中取100μl菌液加入至下一个含有900μl无菌PBS的EP管中,依次稀释,直至获得1×108倍稀释的菌液。
分别从1×106,1×107,1×108倍稀释的菌液中取100μl菌悬液滴加于无菌固体平板中,用玻璃涂布棒将其均匀涂布后用封口膜密封,于严格厌氧环境中倒置培养,每个稀释倍数分别涂布3块平板。待菌落长出后将可以计数的平板(菌落不密集,可清晰分辨)进行计数,对同稀释倍数的3块平板的菌落数取平均值,即为每100μl原菌液在该稀释倍数下所含有的菌落数量,以CFU(colony-forming units,菌落形成单位)表示,最后将平均值乘以稀释倍数即得到每100μl原菌液的菌落数量,本实验中,1×108倍稀释的平板菌落分布均匀,清晰可辨,三次重复计数分别为214、237、223,平均数为228个菌落,经计算得原菌液的菌落数为2.28×1011CFU/ml。
培养后根据计数结果将含菌悬浮液以9000rpm离心10分钟,用无菌PBS重悬菌体两次,最后用含有10%甘油的无菌PBS重悬后以5×1010CFU/ml浓度保存于-80℃。
实施例2
1.小鼠(C57BL/6)心肌梗死模型构建:通过结扎小鼠心脏冠状动脉左前降支来构建心肌梗死小鼠模型。具体的,首先采用质量百分比为4%的水合氯醛溶液以10μl/g的剂量通过腹腔注射麻醉小鼠。用脱毛膏去除其腹侧颈部至剑突之间的毛发。用显微剪剪开颈正中皮肤,暴露出气管,通过留置针将呼吸机导管插入气管中并设置频率每分钟120次。之后在体视镜(购于麦克奥迪医疗诊断系统有限公司)下使用显微剪和显微镊将小鼠的左胸部皮肤及肌肉依次横向剪开约0.8cm小口,在第四肋间用显微镊钝性分离肌肉后暴露出心脏,再用7-0带线缝合针结扎冠状动脉左前降支。最后用5-0缝合线及缝合针依次缝合肋骨、肌肉和皮肤,并用碘伏对手术部位进行消毒。将术后处于麻醉状态的小鼠置于37℃恒温电热毯上,待其恢复行动能力后于原环境中继续饲养。8周后,通过小动物心脏超声成像系统检测心肌梗死小鼠心脏收缩功能,心肌梗死小鼠左心室射血分数与左心室短轴缩短率下降表明心肌梗死模型构建成功。
2.心肌梗死小鼠的抗生素(ABX)处理,具体方法如下:
首先配制ABX,配方如表2所示。
表2抗生素(ABX)的组成
避光条件下称取4种抗生素,在超净工作台中用灭菌双蒸水溶解4种抗生素粉末,配制成ABX溶液,将ABX溶液装入灭菌后的小鼠专用饮水瓶中并用铝箔纸包裹进行避光处理,每2天配制一次ABX溶液防止水体变质。用ABX溶液代替小鼠的日常饮水,共处理7天。7天后小鼠饮用正常的灭菌水。
3.Faecalibacterium prausnitzii的移植
实验分组分为3个组,分别为无菌PBS组,Faecalibacterium prausnitzii(ATCC27768)移植组,灭活组,3个组所用小鼠均是上述经ABX处理后的心肌梗死小鼠。在ABX处理结束后的第1天开始,采用一次性1ml无菌注射器和8号弯头灌胃针通过灌胃的方式对小鼠进行处理,具体方法如下:
第1组PBS组,均按照200μl/只小鼠,用无菌PBS进行灌胃,1周3次,持续8周;
第2组Faecalibacterium prausnitzii(ATCC 27768)移植组,将实施例1提及的保存的5×1010CFU/ml的菌液用无菌PBS稀释至5×108CFU/100μl PBS的浓度后对小鼠进行灌胃,1周3次,持续8周;
第3组灭活组,将5×108CFU/100μl PBS浓度的Faecalibacterium prausnitzii菌液于0.1Mpa 121℃下高温灭菌20分钟,冷却后对灭活组的小鼠进行灌胃,1周3次,持续8周。
8周后实验结束,采用小动物心脏超声成像系统(Vevo2100,Visual Sonics)检测其心脏收缩功能后,安乐死小鼠,解剖后取心脏。然后对心脏样本进行冰冻组织切片,WGA染色(图2),检测心肌肌纤维横截面积的变化,染色结果及统计结果显示,Faecalibacteriumprausnitzii回补减小了心肌梗死小鼠的心肌肌纤维横截面积,而灭活组不具有减小面积的作用。对心脏样本进行石蜡包埋和切片,进行HE染色(图3),检测心肌肌纤维横截面积的变化,染色结果及统计结果显示,Faecalibacterium prausnitzii回补减小了心肌梗死小鼠的心肌肌纤维横截面积,而灭活组不具有减小面积的作用。马松染色(图4),检测心脏纤维组织的面积,染色结果及统计结果显示,Faecalibacterium prausnitzii回补减小了心肌梗死小鼠心脏纤维化的面积,而灭活组不具有减小心脏纤维化面积的作用。
实施例3
1.小鼠心脏超声检测
对小鼠胸部进行脱毛并擦拭干净后,用异氟烷麻醉小鼠,其心率稳定在每分钟450-500次。使用小动物心脏超声检测系统(Visual Sonics,Vevo 2100)检测小鼠心脏收缩功能,采集小鼠左心室B-mode长轴图像并在左心室直径最大处采集M-mode图像。最后用LVtrace工具计算左心室射血分数(EF)、左心室短轴缩短率(FS)。
2.小鼠心肌组织HE染色及马松染色
小鼠心肌组织HE染色:将石蜡切片依次按时放入下列试剂中,对切片进行脱蜡及水化。
自来水冲洗15分钟。之后采用HE染色试剂盒(凯基Cat:KGA224)对切片进行染色,步骤如下:
将切片自然晾干后,用中性树胶及盖玻片封片。将载玻片置于正置显微镜(NiKoneclipse 80i)下观察,心肌组织细胞质呈红色,细胞核呈紫蓝色,红细胞呈桔红色,其他成分呈深浅不同红色。通过NIS-Elements BR软件采集图像并用ImageJ进行测量心肌细胞横截面积。染色结果及统计结果显示,Faecalibacterium prausnitzii回补减小了心肌梗死小鼠的心肌肌纤维横截面积,而灭活组不具有减小面积的作用。
小鼠心肌组织马松染色:将石蜡切片依次按时放入下列试剂中,对切片进行脱蜡及水化:
自来水冲洗15分钟。采用马松三色法染色试剂盒(Servicebio,Cat:G1006)对切片进行染色。步骤如下:
将切片自然晾干后,用中性树胶及盖玻片封片。将载玻片置于正置显微镜(NiKoneclipse 80i)下观察,胶原纤维为蓝色,心肌组织细胞质呈红色,细胞核呈蓝黑色。通过NIS-Elements BR软件采集图像并用ImageJ进行计算胶原纤维面积及心肌组织面积,最后计算得到胶原纤维面积百分比。染色结果及统计结果显示,Faecalibacteriumprausnitzii回补减小了心肌梗死小鼠心脏纤维化的面积,而灭活组不具有减小心脏纤维化面积的作用。
3.小鼠心肌组织的WGA染色
小鼠心肌组织经冰冻切片后产生的组织切片粘附于载玻片上,将载玻片保存于-80℃。具体染色方法如下:
避光条件下用50%封片后,于荧光显微镜(Carl Zeiss Microscopy GmbH)下观察(Hoechst激发波长为375nm,对应的发射波长425nm,以蓝光表示;WGA-FITC激发波长485nm,发射波长525nm,以绿光表示),通过ZEN软件采集图像并用ImageJ测量心肌细胞横截面积。染色结果及统计结果显示,Faecalibacterium prausnitzii回补减小了心肌梗死小鼠的心肌肌纤维横截面积,而灭活组不具有减小面积的作用。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (6)
1.普拉梭菌(Faecalibacterium prausnitzii)在制备治疗心肌梗死后的心室病理性重构的药物中的应用;
所述药物的剂型为液体制剂或固体制剂;
当所述药物的剂型为液体制剂时,所述液体制剂的活菌浓度为109~1012CFU/ml;
所述普拉梭菌为普拉梭菌VPIC13-51,所述普拉梭菌VPIC13-51的ATCC保藏号为ATCC27768。
2.根据权利要求1所述的应用,其特征在于,所述普拉梭菌用改良强化梭菌培养基于严格厌氧环境中培养获得;
所述改良强化梭菌培养基的成分组成为:胰蛋白胨10.0g/L,牛肉膏10.0g/L,酵母提取物3.0g/L,葡萄糖5.0g/L,氯化钠5.0g/L,可溶淀粉1.0g/L,L-半胱氨酸盐酸盐0.5g/L,0.025%的刃天青4ml/L,去离子水1000ml和琼脂15g/L;所述改良强化梭菌培养基的pH值为6.8。
3.根据权利要求1所述的应用,其特征在于,所述液体制剂的活菌浓度为1010~1011CFU/ml。
4.根据权利要求1所述的应用,其特征在于,所述药物通过将所述普拉梭菌菌体悬浮于磷酸盐缓冲液获得。
5.根据权利要求4所述的应用,其特征在于,所述磷酸盐缓冲液还包括体积百分含量为10%的甘油。
6.根据权利要求1所述的应用,其特征在于,所述药物为口服制剂。
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