CN112353822A - 一种乳酸乳球菌粒子与益生菌在制备抗幽门螺旋杆菌感染药物中的应用 - Google Patents
一种乳酸乳球菌粒子与益生菌在制备抗幽门螺旋杆菌感染药物中的应用 Download PDFInfo
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Abstract
本发明涉及生物医药领域，提供了一种乳酸乳球菌粒子与益生菌在制备抗幽门螺旋杆菌感染药物中的应用；所述乳酸乳球菌粒子为乳酸乳球菌NZ9000细胞壁的主要成分肽聚糖，即革兰氏阳性增强子基质颗粒GEM；本发明研究发现，GEM与益生菌制剂（含长双歧杆菌冻干粉、乳双歧杆菌冻干粉、青春双歧杆菌冻干粉等）联用能够有效抑制幽门螺旋杆菌在胃部定植，增加胃部抗菌蛋白的分泌，缓解胃部炎症并进一步增强机体适应性免疫，在治疗由幽门螺旋杆菌感染所致胃部疾病方面具有较大的应用潜力。
Description
技术领域
本发明涉及生物医药领域，尤其涉及一种乳酸乳球菌粒子与益生菌在制备抗幽门螺旋杆菌感染药物中的应用。
背景技术
幽门螺旋杆菌(Helicobacter pylori，Hp)与慢性胃炎、消化性溃疡、胃癌和胃黏膜相关淋巴样组织淋巴瘤等疾病密切相关，在世界卫生组织国际癌症研究机构公布的致癌物清单中，Hp被列为Ⅰ类致癌原。对于Hp的一线治疗方案，从三联疗法发展到今天的四联疗法，都不可避免的需要使用抗生素，而抗生素疗法往往易引起胃肠道菌群失调，导致胃肠功能紊乱等不良反应，同时抗生素的使用也诱导了耐药菌的产生。
相比于抗生素，益生菌在Hp治疗中的优势明显。有研究表明，益生菌具有加强胃黏膜生物屏障、释放细胞毒素及产生短链脂肪酸来抵御Hp感染、竞争抑制Hp定植、平衡多种细胞因子水平等功能。
本发明研究发现来源于乳酸乳球菌（Lactococcus lactis，L. lactis）细胞壁的主要成分为肽聚糖的革兰氏阳性增强子基质颗粒（Gram-positive enhancer matrixparticles，GEMs）能够通过增加 CD4 + T 细胞的募集及抗菌蛋白的产生，而关于本发明的乳酸乳球菌粒子GEM及与益生菌制剂联用目前尚未见报道。
发明内容
本发明基于大量的实验研究和分析，提供了一种乳酸乳球菌粒子GEM与益生菌制剂联用的方式减少幽门螺旋杆菌感染，并通过动物实验验证了其对Hp感染引起的胃炎的治疗效果。
一种抗幽门螺旋杆菌感染的乳酸乳球菌粒子，其特征在于，该乳酸乳球菌菌株为L. lactis NZ9000。
所述的乳酸乳球菌粒子，其特征在于，所述粒子为乳酸乳球菌NZ9000细胞壁的主要成分肽聚糖，即革兰氏阳性增强子基质颗粒。
乳酸乳球菌粒子与益生菌制剂联用在制备抗幽门螺旋杆菌感染药物中的应用。
所述的应用，其特征在于，所述益生菌制剂为衡欣益生菌粉，成分为长双歧杆菌冻干粉、乳双歧杆菌冻干粉、青春双歧杆菌冻干粉、嗜热链球菌冻干粉、嗜酸乳杆菌冻干粉、或德氏乳杆菌保加利亚亚种冻干粉。
所述幽门螺旋杆菌感染所致疾病为慢性胃炎，但不仅限于此。
有益效果：
本发明提供乳酸乳球菌粒子GEM及其与益生菌制剂联用在抗幽门螺旋杆菌感染中的应用。本发明研究发现，GEM与益生菌制剂联用时，可以显著减少Hp的定植，增加胃部抗菌蛋白的表达，缓解炎症，尤其在增强胃部适应性免疫方面，两者联用具有很好的协同作用，能有效治疗幽门螺杆菌感染导致的胃炎等疾病。
附图说明：
图1：肠道菌群16S rRNA基因测序结果；图中，A是肠道菌群主成分分析结果，B是肠道菌群α多样性分析结果，C肠道菌群物种分析结果；NC：空白对照组 HP：HP感染不给药组 K：抗生素给药组 G：GEM给药组 Y：益生菌制剂给药组 GY：GEM+益生菌制剂给药组；
图2：粪便样品中短链脂肪酸含量检测结果；图中，乙酸（A）、丙酸（B）、异丁酸（C）；HP：幽门螺旋杆菌感染组； Antibiotics：抗生素（ 阿莫西林、奥美拉唑、枸橼酸铋钾、甲硝唑）给药组；GEM：GEM给药组；Probiotics：益生菌制剂给药组；GEM+Probiotics：GEM+益生菌制剂给药组；图中，*：P<0.05；**：P<0.01；***：P<0.001；
图3：各组小鼠胃部Hp定植情况结果；
图4：各组小鼠胃部组织HE染色结果；
图5：各组小鼠胃部抗菌蛋白MUC1（A）、S100A8、Angiogenin-4、RegIIIγ（B）的表达情况；
图6：各组小鼠胃部Th1、Th17细胞的分化情况；图中，A是Th1、Th17细胞流式检测结果图，B是Th1细胞流式检测结果柱状图，C是Th17细胞流式检测结果柱状图；
具体实施方式
实施例1
乳酸乳球菌粒子GEM的制备
1、乳酸乳球菌L. lactis NZ9000的培养
种子液培养： M17肉汤培养基置于灭菌锅，115℃灭菌15min，冷却之后的培养基在超净台中加入0.5 %的葡萄糖，取20ml该培养基，加入500µl菌种，30℃静置培养12 h。
扩大培养：分别取1ml种子液，加入两瓶300ml的培养基中，30℃静置培养24 h（瓶底呈白色即乳酸乳球菌）。
2、GEM的制备
①将培养好的乳酸乳球菌菌液4℃、8000r离心10 min，弃上清。
②所得菌体加入100ml 10%三氯乙酸溶液重悬，沸水浴加热30 min（不时搅拌，防止爆沸）。
③加入300 mL PBS搅拌均匀，4℃、8000r离心10 min，弃上清。
④重复③三次以保证三氯乙酸清洗干净，即得GEM。
⑤制备得GEM以PBS重悬，进行真空冷冻干燥，得GEM冻干粉，-20℃保存。
实施例2
GEM与益生菌制剂联用抗幽门螺旋杆菌感染的效果
选取健康成熟的SPF级C57/BL6雌性小鼠，体重18-22g，购自南京青龙山动物繁殖场，共36只，随机分成6组，每组6只，所有小鼠实验前适应性饲养1周。
1、动物分组及给药
Hp感染组（HP组）：每只小鼠每天灌胃200μL生理盐水，共2周，从第3周开始每只小鼠灌胃200μL含1×10 9CFU的Hp的培养基溶液，1周3次（小鼠在灌胃前4 h开始禁食禁水，灌胃后两个小时正常供食供水），3天后处死。
抗生素组（Antibiotics组）：每只小鼠每天四联抗生素给药（80 mg/kg阿莫西林；10 mg/kg奥美拉唑；30 mg/kg枸橼酸铋钾；80 mg/kg甲硝唑），共2周，从第3周开始同上灌胃Hp，3天后处死。
GEM组（GEM组）：每只小鼠灌胃200μL GEM冻干粉溶液（500mg/kg GEM冻干粉），1周1次，共2周，从第3周开始同上灌胃Hp，3天后处死。
益生菌制剂组（Probiotics组）：每天每只小鼠灌胃200μL益生菌制剂溶液（含0.2g益生菌粉末），共2周，从第3周开始同上灌胃Hp，3天后处死。
GEM+益生菌制剂组（GEM+Probiotics组）：小鼠同时灌胃益生菌制剂溶液、GEM冻干粉溶液，用量、方式同上，共2周，从第3周开始同上灌胃Hp，3天后处死。
2、肠道菌群组成变化情况
实验方法：取粪便样品进行肠道菌群16S rRNA基因测序并进行肠道菌群主成分分析、多样性分析、物种组成分析。
实验结果：如图1A所示，第一主成分（PC1）的贡献率为 35 %，在第一主成分上GY组与NC组的交叠程度最大，说明GY给药能够有效减少Hp感染引起的小鼠肠道菌群结构的变化；如图1B所示，多样性指数结果显示：相比其他组小鼠肠道菌群多样性降低，GY组的肠道菌群多样性增加；如图1C所示，与NC组相比，各组的菌群丰度减小，而GY组各菌属的相对丰度有所增加，菌群的组成结构与NC组相似。以上结果说明，联合给药能够有效维持小鼠肠道菌群稳态。
3、肠道菌群的主要代谢产物短链脂肪酸含量检测
实验方法：取粪便样品，气相色谱检测粪便样品中短链脂肪酸含量。
实验结果：如图2所示， 肠道菌群的主要代谢产物乙酸、丙酸、异丁酸的表达量GEM组最高。
4、胃部Hp定植情况
实验方法：取部分胃组织，提取组织RNA并逆转录，通过qPCR检测各组小鼠胃部Hp定植情况。
实验结果：如图1所示，GEM单用或与益生菌制剂联用能够显著减少Hp的定植。
5、胃部炎症情况
实验方法：取部分胃组织，用4％多聚甲醛固定，再进行石蜡包埋、切片、HE染色，观察炎症情况。
实验结果：如图2所示，Hp感染导致小鼠胃部组织中出现腺窝缺失及大量炎症细胞浸润，抗生素给药使得炎症症状加重，GEM组、益生菌制剂组炎症缓解，其中，GEM与益生菌制剂联用具有协同作用，炎症缓解效果最好。
6、胃部抗菌蛋白表达情况
实验方法：取部分胃组织，提取组织RNA并逆转录，通过qPCR检测各组小鼠胃部抗菌蛋白MUC1、S100A8、Angiogenin-4、RegIIIγ的表达情况。
实验结果：如图3所示，抗生素组和益生菌制剂组效果相近，GEM组和联用组小鼠胃部抗菌蛋白的表达明显高于这两组，说明GEM单用或与益生菌制剂联用能够显著增加抗菌蛋白的分泌，从而抑制Hp在胃部的定植。
7、胃部Th1、Th17细胞的分化情况
实验方法：取胃组织，通过密度梯度离心提取淋巴细胞，体外刺激其分泌细胞因子并阻断细胞因子分泌到胞外，然后流式检测各组样品Th1、Th17细胞的表达情况。
实验结果：如图4所示，GEM与益生菌制剂联用时，Th1、Th17细胞的比例明显高于GEM组、益生菌制剂组，说明两者联用具有协同作用，能够进一步增强小鼠的胃部适应性免疫，抵抗Hp感染。
Claims (3)
1.一种乳酸乳球菌粒子与益生菌在制备抗幽门螺旋杆菌感染药物中的应用，其特征在于，该乳酸乳球菌菌株为L. lactis NZ9000。
2.如权利要求1所述的应用，其特征在于，所述粒子为乳酸乳球菌NZ9000细胞壁的主要成分肽聚糖，即革兰氏阳性增强子基质颗粒。
3.如权利要求1或2所述的应用，其特征在于，所述益生菌制剂为衡欣益生菌粉，成分为长双歧杆菌冻干粉、乳双歧杆菌冻干粉、青春双歧杆菌冻干粉、嗜热链球菌冻干粉、嗜酸乳杆菌冻干粉、或德氏乳杆菌保加利亚亚种冻干粉。
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