CN114437996B - 一种用于预防或治疗哮喘的热灭活酪酸梭菌及其制备方法 - Google Patents
一种用于预防或治疗哮喘的热灭活酪酸梭菌及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于预防或治疗哮喘的热灭活酪酸梭菌及其制备方法,属于医药技术领域。所述的热灭活酪酸梭菌是将酪酸梭菌菌粉置于110kpa和121℃高温高压热灭活20分钟获得。本发明的热灭活酪酸梭菌在哮喘小鼠模型实验中可较好的抑制气道炎症和气道黏液分泌的能力,并有免疫调节功能,能够有效防治哮喘,还可用于其他过敏性疾病的预防或治疗,具有良好的应用前景。
Description
技术领域
本发明涉及一种用于预防或治疗哮喘的热灭活酪酸梭菌及其制备方法,属于医药技术领域。
背景技术
哮喘是常见的慢性呼吸道疾病,全球约有3亿哮喘患者,我国20岁以上的哮喘患者达4000多万,其发病率在低中收入国家仍呈上升趋势。哮喘反复发作严重影响患者的学习、工作、社会生活和体育活动,每年的直接医疗支出及旷工、死亡造成的经济损失不计其数,是全球重大的卫生健康问题。
目前吸入性糖皮质激素、β肾上腺素受体激动剂、白三烯受体拮抗剂和茶碱等常用哮喘治疗药物只能控制症状,并不能根治哮喘。这些药物治疗时间长,具有一定的副作用,依从性不佳,并且有部分患者对这些药物不敏感。因此,急需寻找有效的哮喘防治新药。
益生菌是一类摄入适当的量对宿主健康带来益处的微生物。益生菌具有调节免疫、抗肿瘤、抗感染、抗氧化、抗衰老等功效,已广泛应用于畜牧养殖、食品添加和医疗保健,但其安全性正受到越来越多的关注。益生菌能够平衡人体菌群结构,促进抗原降解,调节促炎和抗炎因子等免疫功能。由于益生菌具有重要的免疫调节作用,因此通过益生菌治疗哮喘等过敏性疾病成为近年来研究的热点。目前用于治疗哮喘研究的益生菌主要包括加氏乳杆菌A5(Lactobacillus gasseri A5)、嗜酸乳杆菌、双歧杆菌、鼠李糖乳杆菌、副干酪乳杆菌等。
酪酸梭菌(拉丁学名:Clostridium butyricum),又称:酪酸梭状芽孢杆菌,酪酸杆菌,酪酸菌,丁酸梭状芽孢杆菌,丁酸梭菌,丁酸杆菌,丁酸菌,是临床常用的益生菌,已有研究报道,酪酸梭菌具有抗哮喘功效,可用于哮喘的预防或治疗。酪酸梭菌和热灭活的酪酸梭菌均具有免疫调节功能,但热灭活的酪酸梭菌是否有抗哮喘功效尚不清楚,目前尚未见有关于热灭活酪酸梭菌在防治哮喘方面的相关研究报道。
发明内容
本发明的目的在于提供一种用于预防或治疗哮喘的热灭活酪酸梭菌及其制备方法。
为实现上述目的,本发明采用如下技术方案:
一种用于预防或治疗哮喘的热灭活酪酸梭菌,其制备方法为:先测定酪酸梭菌菌粉的含菌量,再称取一定重量的酪酸梭菌菌粉置于110kpa和121℃高温高压热灭活20分钟获得热灭活酪酸梭菌。
上述的用于预防或治疗哮喘的热灭活酪酸梭菌的制备方法所制备得到的热灭活酪酸梭菌,自然冷却至室温后保存于-20℃的温度下,使用前,热灭活的酪酸梭菌加入磷酸盐缓冲液(PBS),将其配制成含菌量(按未灭活前的含菌量算)为5X108CFU/ml的混悬液;超声细胞破碎仪40%的功率破碎4次促进溶解,每次超声持续时间15s,间隔20s;然后再加入PBS将其配成使用所需浓度。
进一步的,上述的用于预防或治疗哮喘的热灭活酪酸梭菌,所用于热灭活的酪酸梭菌选用保藏于中国普通微生物菌种保藏管理中心,保藏编号为:CGMCC NO:0313-1的酪酸梭菌。
上述的用于预防或治疗哮喘的热灭活酪酸梭菌,所述的热灭活酪酸梭菌在制备用于预防或治疗哮喘的药物中的用途。所述的热灭活酪酸梭菌可以作为单独制剂,也可以和其它药物配伍,加入适量药剂学上可接受的载体,采用现有技术制成可药用的剂型。如气雾剂、吸入剂、口服液、颗粒剂等。
本发明考察了雾化吸入和口服热灭活酪酸梭菌的抗哮喘作用,发明人发现,本发明的热灭活酪酸梭菌不仅能够有效防治哮喘,而且其具有免疫调节功能,还可用于其他过敏性疾病的预防或治疗。
本发明的有益效果:
1、本发明通过试验证明:雾化吸入和口服本发明的热灭活酪酸梭菌均可减少哮喘小鼠的气道炎症和黏液分泌;雾化吸入热灭活酪酸梭菌具有免疫调节功能,可通过抑制哮喘小鼠肺泡灌洗液IL-4的含量、脾脏Th17细胞的比例和Th17/Treg细胞的比值,增加肺泡灌洗液IFN-γ的含量和脾脏Treg细胞的比例发挥其抗哮喘作用。本发明提供的热灭活酪酸梭菌能够有效防治哮喘,可用于制备预防或治疗哮喘的药物。
2、本发明提供了一种用于预防或治疗哮喘的热灭活酪酸梭菌及其制备方法,为临床防治哮喘提供了新的途径和方案。而且相比于酪酸梭菌活菌,本发明提供的热灭活酪酸梭菌治疗哮喘的效果更加突出,具有显著性进步。此外,相比于酪酸梭菌活菌,热灭活的酪酸梭菌还具有安全可靠,无感染风险;稳定性佳,可标准化生产,存储方便,使用便捷,生物效价高等优点,值得进一步临床推广应用。
附图说明
图1肺泡灌洗液炎症细胞总数和嗜酸性粒细胞比例;
图2肺组织HE染色;
图3肺组织PAS染色;
图4肺泡灌洗液IFN-γ和IL-4的浓度水平。
具体实施方式
1.材料与方法
1.1材料:酪酸梭菌CGMCC NO:0313-1由山东科兴生物制药股份有限公司提供;
1.2实验方法
1.2.1热灭活酪酸梭菌:先测定酪酸梭菌菌粉的含菌量,再称取一定重量的酪酸梭菌菌粉置于110kpa和121℃高温高压热灭活20分钟获得热灭活酪酸梭菌。自然冷却至室温后保存于-20℃。
1.2.2热灭活酪酸梭菌菌液的准备:使用前,热灭活的酪酸梭菌加入磷酸盐缓冲液(PBS),将其配制成含菌量(未灭活前的含菌量)为5X108CFU/ml的混悬液。超声细胞破碎仪40%的功率破碎4次促进溶解,每次超声持续时间15s,间隔20s。然后再加入PBS将其配成使用所需浓度。
1.2.3实验动物分组:64只BALB/c雄性小鼠随机分为8组,每组8只,分别为正常对照组(A组)、哮喘对照组(B组)、雾化吸入低剂量热灭活酪酸梭菌干预组(C组)、雾化吸入高剂量热灭活酪酸梭菌干预组(D组)、口服低剂量热灭活酪酸梭菌干预组(E组)、口服高剂量热灭活酪酸梭菌干预组(F组)、口服低剂量酪酸梭菌活菌干预组(G)和口服高剂量酪酸梭菌活菌干预组(H组)。
1.2.4哮喘模型的建立:采用卵清蛋白(OVA)致敏法,于实验第0、7、14天小鼠腹腔注射致敏液0.2ml(25μg OVA+1mg氢氧化铝+200μl PBS液)。第21天起,将小鼠置于30cm X20cm X 30cm雾化箱内,雾化吸入2%OVA激发,每次30min,每天1次,连续7天,建立哮喘模型。
1.2.5各组小鼠的干预:B、C、D、E、F、G和H组按上述方法建立哮喘模型。A组以PBS替代OVA腹腔注射和雾化。C、D、E、F、G和H组在实验第0-13天进行药物干预,每天1次,连续14天。
药物干预处理如下:C组予含1X107CFU的热灭活酪酸梭菌液20ml雾化吸入,每次雾化30分钟;D组予含1X108CFU的热灭活酪酸梭菌液20ml雾化吸入,每次雾化30分钟;E组每只小鼠每天予含1X107CFU的热灭活酪酸梭菌液0.2ml灌胃;F组每只小鼠每天予含1X108CFU的热灭活酪酸梭菌液0.2ml灌胃;G组每只小鼠每天予含1X107CFU的酪酸梭菌活菌液0.2ml灌胃;H组每只小鼠每天予含1X108CFU的酪酸梭菌活菌液0.2ml灌胃。
1.2.6BALF炎症细胞和分类计数:末次激发24小时内处死小鼠,BALF行炎症细胞总数和分类计数。
1.2.7肺组织病理染色和评分:左肺组织行HE染色和PAS染色。根据细支气管周围炎症细胞浸润程度进行气道炎症评分:无炎症细胞浸润,0分;少量炎症细胞浸润,1分;有1层炎症细胞浸润,2分;有2-4层炎症细胞浸润,3分;超过4层炎症细胞浸润,4分;根据杯状细胞占细支气管上皮细胞的百分比进行PAS评分:细支气管无杯状细胞,0分;0<杯状细胞比例<25%,1分;25%≤杯状细胞比例≤50%,2分;50%<杯状细胞比例≤75%,3分;75%<杯状细胞比例,4分。
1.2.8ELISA法检测BALF中IFN-γ和IL-4的浓度水平。
1.2.9流式细胞术检测脾Th17和Treg细胞比例。
2.统计学方法:符合正态分布且方差齐的计量资料以表示,组内两两比较使用LSD检验;不符合正态分布的以中位数(四分位间距)表示,使用秩和检验分析。P<0.05为差异有显著性。与A组比较,*P<0.05;与B组比较,#P<0.05。
3.结果
3.1行为症状改变:H组2只小鼠干预过程中出现腹泻,其余5组干预组小鼠未见明显不良反应;B组小鼠激发过程中出现哮喘发作样症状,6组药物干预组哮喘发作样症状均减轻。
3.2BALF炎症细胞总数和细胞分类计数:与A组对比,B组小鼠的炎症细胞总数和嗜酸性粒细胞比例显著增加(P<0.05);与B组对比,6组药物干预组炎症细胞总数和嗜酸性粒细胞比例显著减少(P<0.05),其中雾化吸入高剂量热灭活酪酸梭菌干预组(D组)效果最好,见附图1。
3.3肺组织病理观察:与A组对比,B组小鼠肺组织见气道周围大量的炎症细胞浸润和气道黏液分泌增加,气道炎症评分和PAS评分显著增加(P<0.05);与B组对比,6组药物干预组的气道炎症细胞浸润和气道黏液分泌均减少,气道炎症评分和PAS评分均显著减少(P<0.05),其中雾化吸入高剂量热灭活酪酸梭菌干预组(D组)效果最好,见附图2、3和表1。
表1气道炎症评分和PAS评分M(P25,P75)
3.4BALF中IFN-γ和IL-4浓度水平:与A组对比,B组小鼠的IFN-γ浓度水平显著下降(P<0.05),IL-4浓度水平显著增高(P<0.05);与B组对比,雾化吸入高剂量热灭活酪酸梭菌干预组(D组)的IFN-γ浓度水平显著增高(P<0.05),IL-4浓度水平显著下降(P<0.05),见附图4;
3.5脾脏Th17和Treg细胞的比例:与A组对比,B组小鼠的Th17细胞比例和Th17/Treg比值显著增加(P<0.05);与B组对比,雾化吸入高剂量热灭活酪酸梭菌干预组(D组)的Treg细胞显著增加(P<0.05),Th17细胞比例和Th17/Treg比值显著减少(P<0.05),见表2。
表2脾脏Th17和Treg细胞M(P25,P75)
结论:雾化吸入热灭活酪酸梭菌、口服热灭活酪酸梭菌和口服酪酸梭菌活菌均可显著减少哮喘小鼠的气道炎症和气道黏液分泌,其中雾化吸入热灭活酪酸梭菌的效果最好;雾化吸入热灭活酪酸梭菌可通过调节免疫细胞和细胞因子发挥抗哮喘作用;雾化吸入和口服热灭活酪酸梭菌均可应用于哮喘的预防或治疗。
Claims (2)
1.热灭活酪酸梭菌菌液在制备用于预防或治疗哮喘的药物中的用途,所述的热灭活酪酸梭菌菌液的制备方法为:先测定酪酸梭菌菌粉的含菌量,再称取一定重量的酪酸梭菌菌粉置于110kpa和121℃高温高压热灭活20分钟,得到热灭活酪酸梭菌,自然冷却至室温后保存于-20℃的温度下,使用前,热灭活酪酸梭菌加入磷酸盐缓冲液,将其配制成含菌量,按未灭活前的含菌量算,为5×108CFU/ml的混悬液;超声细胞破碎仪40%的功率破碎4次促进溶解,每次超声持续时间15s,间隔20s;然后再加入磷酸盐缓冲液将其配成使用所需浓度;所用于热灭活的酪酸梭菌选用保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCC NO:0313-1的酪酸梭菌。
2.根据权利要求1所述的热灭活酪酸梭菌菌液在制备用于预防或治疗哮喘的药物中的用途,其特征在于,所述的热灭活酪酸梭菌菌液单独制剂,或者和其它药物配伍,加入适量药剂学上可接受的载体,采用现有技术制成可药用的剂型。
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