CN117089490B - 青春双歧杆菌basj001及应用 - Google Patents
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Abstract
本发明提供青春双歧杆菌BASJ001在免疫检查点抑制剂抗PD‑1抗体的增敏剂制备中的应用以及青春双歧杆菌BASJ001在缓解免疫检查点抑制剂抗PD‑1抗体所产生的免疫治疗相关性不良反应的药物制备中的应用，青春双歧杆菌BASJ001，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.26098。本发明减少血清中AST水平，减轻免疫治疗相关性肠炎和肝损伤，在临床上合理应用可有效避免或减轻副反应；本发明可以为今后探究增敏免疫治疗疗效的方法提供了新线索和新思路。
Description
技术领域
本发明属于益生菌技术领域，具体为青春双歧杆菌BASJ001及应用。
背景技术
肿瘤免疫治疗可重启机体正常免疫反应，减少肿瘤免疫逃逸和免疫耐受，是癌症治疗的一项重大突破。多种免疫检查点抑制剂(ICIs)药物如程序性细胞死亡蛋白-1(PD-1)及其配体PD-L1抗体已应用于临床肿瘤免疫治疗。然而有限的反应率、缺乏预测临床反应的标志物以及潜在的毒副作用，阻碍了免疫治疗的进一步应用。因此，寻找安全有效的增强免疫治疗的方式显得尤为重要。
越来越多的证据表明，肠道微生物不仅可以通过调节免疫直接影响肿瘤的发生发展，并且在免疫治疗中发挥着重要作用，肠道微生物与免疫治疗的反应性密切相关。随着技术的进展，大量肠道微生物被发现能够通过抗原模拟、代谢物的作用、肽聚糖、胞外多糖等起到增强免疫治疗的作用。此外，研究表明，双歧杆菌属与免疫治疗的敏感性密切相关。
青春双歧杆菌(Bifidobacterium adolescentis)是一种完全厌氧的革兰氏阳性菌，可以维持肠内菌群平衡，调节人体肠道稳态。青春双歧杆菌已经在多个方面证明了其有益功能，并在食品和药品中得到应用，如改善急慢性腹泻、便秘、炎症性肠病以及延缓衰老等。然而目前尚未有青春双歧杆菌与改善肿瘤免疫治疗疗效的相关性研究。
发明内容
本发明第一个目的在于，提供青春双歧杆菌BASJ001的一种新应用。
为此，本发明的上述目的通过如下技术方案实现：
青春双歧杆菌BASJ001在免疫检查点抑制剂抗PD-1抗体的增敏剂制备中的应用，青春双歧杆菌BASJ001，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CGMCCNO.26098。
青春双歧杆菌BASJ001，由健康成年人的粪便中分离所得，命名为青春双歧杆菌BASJ001，保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址为北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC NO.26098，保藏日期2022年11月10日。
青春双歧杆菌(Bifidobacterium adolescentis)BASJ001，是从浙江健康成年人粪便样本中分离得到的。该菌株经16S rDNA序列分析，并在NCBI中进行核酸序列比对，发现其与青春双歧杆菌的核酸序列相似度最高(近100％)，提示该菌株属于双歧杆菌属的青春双歧杆菌。将上述菌株进行全基因组测序并与NCBI所有已公布全基因组序列的其它青春双歧杆菌株进行泛基因组比对分析，发现该菌株与已公布的青春双歧杆菌菌株的基因组均有明显不同，鉴定为新的青春双歧杆菌株。
本发明还有一个目的在于，提供青春双歧杆菌BASJ001的另一种新应用。
为此，本发明的上述目的通过如下技术方案实现：
青春双歧杆菌BASJ001在缓解免疫检查点抑制剂抗PD-1抗体所产生的免疫治疗相关性不良反应的药物制备中的应用，青春双歧杆菌BASJ001，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.26098。
本发明提供青春双歧杆菌BASJ001的新应用，与现有技术相比，至少具有如下优点及有益效果：
(1)、青春双歧杆菌BASJ001可以改善肿瘤免疫治疗疗效，目前尚无相关文献和专利报道，可作为当今有限益生菌治疗手段的有益补充；
(2)、青春双歧杆菌BASJ001可以通过增加肿瘤中CD8+T细胞TNF-α的分泌增强肿瘤的杀伤作用；
(3)、青春双歧杆菌BASJ001可以减少结肠中IL-1β、IL-17a、IFN-γ等炎性因子分泌，减少血清中AST水平，减轻免疫治疗相关性肠炎和肝损伤，在临床上合理应用可有效避免或减轻副反应；
(4)、本发明可以为今后探究增敏免疫治疗疗效的方法提供了新线索和新思路。
附图说明
图1为实施例1中青春双歧杆菌BASJ001的菌落照片。
图2a为BASJ001菌的Circos基因组圈图分析；图2b为BASJ001菌亚种比较基因组物种进化树分析；图2c为BASJ001菌亚种泛基因组Venn图分析。
图3为实施例2中青春双歧杆菌BASJ001联合抗PD-1抗体对小鼠结直肠肿瘤的影响图示；图中分别表示PBS联合isotype组、BASJ001联合isotype组、PBS联合抗PD-1抗体组、BASJ001联合抗PD-1抗体组肿瘤重量差异情况。
图4为实施例3中青春双歧杆菌BASJ001联合抗PD-1抗体在对CD8+T细胞中TNF-α分泌的影响图示；图中分别表示PBS联合isotype组、BASJ001联合isotype组、PBS联合抗PD-1抗体组、BASJ001联合抗PD-1抗体组肿瘤中CD8+T细胞分泌TNF-α的数量差异。
图5为实施例4中青春双歧杆菌BASJ001联合抗PD-1抗体对免疫治疗引起的结肠中炎性因子分泌和血清中AST水平的影响图示；(a)为PBS联合抗PD-1抗体组、BASJ001联合抗PD-1抗体组中结肠中炎性因子IL-1β的分泌图示；(b)为PBS联合抗PD-1抗体组、BASJ001联合抗PD-1抗体组中结肠中炎性因子IL-17a的分泌图示；(c)为PBS联合抗PD-1抗体组、BASJ001联合抗PD-1抗体组中结肠中炎性因子IFN-γ的分泌图示；(d)为PBS联合抗PD-1抗体组、BASJ001联合抗PD-1抗体组血清中AST水平图示。
具体实施方式
参照附图和具体实施例对本发明作进一步详细地描述。
实施例1：青春双歧杆菌BASJ001的分离、筛选和鉴定
(1)、菌株分离：在厌氧工作站中，取浙江杭州健康成年人的健康粪便样品2g，用无菌生理盐水经梯度稀释后涂布于RCM培养基平板，37℃厌氧培养。挑选单菌落进行纯化培养。
(2)、菌株富集：将纯化培养后的菌株，在无菌操作台上用接种环逐一接种到无菌RCM肉汤培养基中，置于摇床中，37℃培养48h后进行16S rDNA测序鉴定。
(3)、16S rDNA测序鉴定：使用细菌通用引物对BASJ001菌株进行菌落PCR扩增，送至上海美吉生物公司进行16S rDNA测序鉴定，在NCBI上进行序列比对该菌株16S rDNA的长度为1526bp，序列如下，发现与双歧杆菌属的Bifidobacterium adolescentis 16S rDNA序列同源相似性最高(比对相似度约为100％)，确定该菌株为青春双歧杆菌。
(4)、全基因组鉴定：将上述青春双歧杆菌BASJ001进一步在上海美吉生物公司进行全基因组测序，并对测序结果进行Circos基因组圈图分析、进化树分析以及泛基因组的venn图分析。结果如下：如图2a，为BASJ001菌株的全基因组测序完成图(Circos基因组圈图分析)；如图2b所示，采用MEGA软件，邻位连接法显示“青春双歧杆菌BASJ001菌株”与目前NCBI已公布全基因组序列的所有相关株的16S rDNA序列比较，构建系统发育树，青春双歧杆菌BASJ0001拥有独立的叶节点，与其它已知报道的青春双歧杆菌菌株存在差异；如图2c所示，根据泛基因组分析结果，通过Venn图的形式表示所有检测的菌株样本的核心基因以及特有基因，青春双歧杆菌BASJ001拥有不同于其它已知报道的青春双歧杆菌菌株的特有基因。综上结果以及根据文献调研，该BASJ001菌株不同于既往已报道的菌株，为新发现的青春双歧杆菌菌株。
该菌株已于2022年11月10日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏单位地址：北京市朝阳区北辰西路1号院3号；保藏编号为CGMCC NO.26098，建议的分类学名称：青春双歧杆菌(Bifidobacterium adolescentis)。
实施例2：青春双歧杆菌BASJ001增敏抗PD-1抗体在结直肠癌中的治疗作用
将6周雄性C57BL/6小鼠，随机分为4组，每组10只，分别为：PBS+isotype组、PBS+抗PD-1抗体组、青春双歧杆菌BASJ001+isotype组、青春双歧杆菌BASJ001+抗PD-1抗体组。PBS为青春双歧杆菌BASJ001溶剂，为无青春双歧杆菌BASJ001的溶剂对照。isotype为与抗PD-1抗体相同来源的抗体，但其无抗PD-1活性，为抗PD-1抗体的阴性同型对照。小鼠造模前自由饮用含2mg/mL甲硝唑、2mg/mL青霉素、2mg/mL链霉素和1mg/mL万古霉素的饮水，1周初步清除肠道菌群，有利于移植菌定植。
肠道菌群清除后，青春双歧杆菌BASJ001+isotype组、青春双歧杆菌BASJ001+抗PD-1抗体组给予青春双歧杆菌BASJ001灌胃，剩余两组PBS灌胃，剂量为1×109CFU/只小鼠，灌胃量200μL，每天一次。灌胃7天后，将2×107的MC38细胞注射至小鼠皮下(每只小鼠100μL)。注射10天后，给PBS组和青春双歧杆菌BASJ001组腹腔注射IgG Isotype、PBS+抗PD-1抗体组和青春双歧杆菌BASJ001+抗PD-1抗体组腹腔注射抗PD-1抗体(100μg每只)每隔两天打一针，每两天监测肿瘤体积，计算公式如下：体积＝0.54×L×W2，其中L为最长直径，W为最短直径。三针PD-1治疗后隔天终止实验，记录并统计肿瘤重量。
结果如图3所示，青春双歧杆菌BASJ001+抗PD-1抗体组肿瘤大小明显小于PBS+抗PD-1抗体组，青春双歧杆菌BASJ001显著增加PD-1的治疗作用。
实施例3：青春双歧杆菌BASJ001增加抗PD-1抗体治疗时在结直肠癌肿瘤中CD8+T细胞TNF-α的分泌
取实施例2中四组：PBS+isotype组、PBS+抗PD-1抗体组、青春双歧杆菌BASJ001+isotype组、青春双歧杆菌BASJ001+抗PD-1抗体组的皮下瘤的小鼠模型的小鼠肿瘤组织，去除脂肪组织和系膜，置于预冷1640培养基中，纵向切开，用1640培养基清洗2-3遍，各组截取肿瘤位置及大小尽量保持一致(绿豆大小左右)。将肠道剪碎成肉糜，收集到新的管子中，加入5mL消化液(消化液配方：Hanks溶液+5％灭活FBS+1mg/mL四型胶原酶)，置于37℃摇床中，200rpm 20min。结束后用300目滤膜过滤入15mL离心管，800g，5min离心弃上清。配置Cocktail刺激液(1640培养基+10％血清+1％青链霉素+0.2％cocktail)，将收集的细胞以500μl Cocktail刺激液重悬，放入24孔板在细胞培养箱中37℃孵育4h。4h后收集细胞800g，5min，离心弃上清。而后调整细胞浓度：将收集的单细胞悬液用PBS将细胞数调至2×107/mL。每个样本取50μL细胞悬液加入0.3μL死活抗体后混匀，避光4℃孵育30min，加入150μLFACS buffer终止染色，800g 5min离心，弃上清，50μL PBS重悬。配制抗体预混液(FVS 510、AlexaFluor 700-CD45、PECP-CY5.5-CD3、BV605-CD4、APC-CY7-CD8、PE-CF594-TNF-α、BV421-IFN-γ)加入适量抗体混匀，避光4℃孵育染色30min，加入150μLPBS终止，800g 5min离心，弃上清，加入150μl固定液常温避光固定20min，800g 5min离心，弃上清。加入150μl破膜液重悬，立即800g 5min离心，弃上清，50μl破膜液重悬，配制抗体预混液(FVS 510、AlexaFluor 700-CD45、PECP-CY5.5-CD3、BV605-CD4、APC-CY7-CD8、PE-CF594-TNF-α、BV421-IFN-γ)常温避光染色20min。加入150μl PBS终止，800g 5min离心，弃上清，用150μL PBS洗涤1次，适当体积重悬上机。
结果如图4所示，青春双歧杆菌BASJ001显著增加了PD-1治疗下，小鼠MC38肿瘤模型中，肿瘤组织中CD8+T细胞中TNF-α的分泌，增强了CD8+T细胞的杀伤作用。
实施例4：青春双歧杆菌BASJ001减少免疫治疗引起的结肠中炎性因子的分泌和血清中AST的水平。
取实施例2中两组：PBS+抗PD-1抗体组、青春双歧杆菌BASJ001+抗PD-1抗体组的皮下瘤的小鼠模型，收集小鼠结肠组织中段，提取RNA，进行逆转录获得cDNA，采用实时荧光定量PCR检测小鼠结肠组织中炎性因子iL-1β，iL-17a，IFN-γ的表达水平。收集小鼠血清，用谷草转氨酶比色法测试盒(Elbascience)对血清中谷草转氨酶的含量进行检测。
结果如图5所示，青春双歧杆菌BASJ001显著降低了PD-1治疗下，肠道组织中IL-1β，IL-17a，IFN-γ等炎性因子分泌，并且降低了血清中AST的含量，降低了PD-1引起的相关不良反应。
上述具体实施方式用来解释说明本发明，仅为本发明的优选实施例，而不是对本发明进行限制，在本发明的精神和权利要求的保护范围内，对本发明做出的任何修改、等同替换、改进等，都落入本发明的保护范围。
Claims (2)
1. 青春双歧杆菌BASJ001在免疫检查点抑制剂抗PD-1抗体的增敏剂制备中的应用，青春双歧杆菌BASJ001，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.26098。
2. 青春双歧杆菌BASJ001在缓解免疫检查点抑制剂抗PD-1抗体所产生的免疫治疗相关性不良反应的药物制备中的应用，所述免疫治疗相关性不良反应为免疫治疗相关性结肠炎和肝损伤，青春双歧杆菌BASJ001，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.26098。
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