CN114404457B - 植物乳杆菌l168在制备延缓卵巢衰老的药物中的应用 - Google Patents
植物乳杆菌l168在制备延缓卵巢衰老的药物中的应用 Download PDFInfo
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Abstract
本发明公开了植物乳杆菌L168在制备延缓卵巢衰老的药物中的应用。植物乳杆菌L168能够提高kdm5基因缺失果蝇成熟卵子数量及子代孵化率，提高衰老果蝇导致果蝇成熟卵子数量及子代孵化率，改善了kdm5基因缺失导致卵母细胞提前激活的现象，改善了自然衰老果蝇的滋养细胞提前激活的现象，因此，植物乳杆菌L168能够在制备延缓卵巢衰老的药物中应用。
Description
技术领域
本发明涉及微生物技术领域，涉及植物乳杆菌L168在制备延缓卵巢衰老的药物中的应用。
背景技术
近年来，由于来自社会竞争的加剧，就业压力的加大，以及环境的不断恶化等因素，不孕不育症的发病率已呈极速的上升的趋势。WHO预测其将与心脑血管疾病，肿瘤一同被并入21世纪人类三大疾病。
不孕不育症是男性或女性生殖系统疾病，指一年以上未采取任何避孕措施，性生活正常而没有成功妊娠。它影响着全球数以百万计的育龄人，并对其家庭和社区产生影响。据估算，全球供有4800万对夫妇和1.86亿人患不孕不育症。女性生殖系统不孕由卵巢、子宫、输卵管和内分泌系统等一系列异常引起。
卵巢衰老是不孕不育的一个重要病因，卵巢衰老是指随着年龄增长而导致的女性储备逐渐衰退直至衰竭的过程，以卵泡数量和卵子质量下降为基础，最终表现为绝育乃至绝经，并影响多个系统和器官，导致相关疾病和综合征的发生发展。
因此，鉴于不孕不育症对人们生活质量和福祉的重要性和影响，全球正在积极开展全球不孕不育症流行病和病原学研究。随着肠道菌群研究热度的不断提升，科学们开始意识到，肠道菌群稳态在女性生殖也发挥着重要作用。
肠道菌群常被称为人体中存在的第9大系统以及第2套基因组，肠道中所存在的各种细菌的总含量已经超过了人体的总细胞量，被称为人体的另一个“器官”。此外肠道菌群所编码的基因的总和是人体基因的约150倍左右。肠道菌群参与人体营养物质的转化与吸收，并与身体各组织，器官存在密切的联系，成熟稳定的肠道菌群可以增加肠粘膜稳定性，协助人体能量摄取与代谢，提高免疫系统的发展与成熟，肠道微生态失衡，不仅会产生多种胃肠道疾病，还会影响机体的代谢，内分泌系统和免疫系统，并通过肠-卵巢轴影响生殖细胞发育和成熟。但不同的肠道菌株具备不同的功能，目前尚未见植物乳杆菌能够通过调控卵母细胞的发育影响宿主生殖发育或延缓生殖衰老的相关报道。
发明内容
本发明的目的是针对现有技术的上述不足，提供植物乳杆菌L168在制备延缓卵巢衰老的药物中的应用。
本发明的目的可通过以下技术方案实现：
植物乳杆菌L168在制备延缓卵巢衰老的药物中的应用，所述的植物乳杆菌 L168于2017年2月13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CGMCC，保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCCNo.13660。
有益效果：
植物乳杆菌L168能够提高kdm5基因缺失果蝇成熟卵子数量及子代孵化率，提高衰老果蝇导致果蝇成熟卵子数量及子代孵化率，改善了kdm5基因缺失导致卵母细胞提前激活的现象，改善了自然衰老果蝇的滋养细胞提前激活的现象，因此，植物乳杆菌L168能够在制备延缓卵巢衰老的药物中应用。
附图说明
图1：各组果蝇卵巢DAPI染色图及成熟卵子数量统计(A)、各组果蝇与野生型果蝇W1118雄蝇杂交产卵量(n)及子代孵化率(B)
图2：各组果蝇卵巢DAPI染色图及成熟卵子数量统计(A)、各组果蝇与野生型果蝇W1118雄蝇杂交产卵量(n)及子代孵化率(B)
图3：各组果蝇卵母细胞在减数分裂阻滞期间转录水平(EU水平)及统计
图4：各组果蝇滋养细胞在减数分裂阻滞期间转录水平(EU水平)及统计
生物材料保藏信息
植物乳杆菌(lactobacillus plantarum)L168保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏日期为2017年2月13日,保藏编号为CGMCC No.13660。
具体实施方式
实施例1：果蝇品系及培养方法
果蝇食物配方：蔗糖(31.62g/L)，葡萄糖(63.24g/L),酵母粉(31.19g/L), 玉米粉(77.7g/L),琼脂粉(10.6g/L),无水CaCl2(0.726g),山梨酸钾(2g)。培养方法：本实验果蝇饮食按照上述实验配方，12小时光照黑暗昼夜交替，25 度，70％湿度环境下生长。
果蝇品系：WT1118,kdm510424；KDM5-RNAi(TRIP line 35706),nos-GAL4(theBloomington stock center)
实施例2植物乳杆菌L168菌液的制备方法
配制MRS培养基：称酪蛋白胨10.0克，牛肉浸取物10.0克，酵母提取液 5.0克，葡萄糖5.0克，乙酸钠5.0克，柠檬酸二胺2.0克，吐温80 1.0克，磷酸氢二钾2.0克，七水硫酸镁0.2克，七水硫酸锰0.05克，碳酸钙20.0克，蒸馏水 1.0升，pH 6.8。按1：500比例将菌种种于MRS液体培养基中，于37℃厌氧培养箱培养，根据细菌吸光度(OD600)及细菌平板计数，确定细菌浓度。培养至对数期末期，取出菌液，12000rpm/min离心15-20min，用PBS洗涤2-3次后，用PBS将浓度调整至1x108 CFU/ml，在果蝇食物上铺一层滤纸并滴入100ul菌液喂食果蝇。
实施例3植物乳杆菌L168提高kdm5基因缺失果蝇成熟卵子数量及子代孵化率
3.1实验方法
3.1.1分组信息
对照组(nos-gal4；GFP RNAi)：羽化3天的野生型果蝇
对照治疗组组(nos-gal4；GFP RNAi+LP168)：刚羽化的新生野生型果蝇，每天一次喂食新鲜的，浓度为108CFU/ml的植物乳杆菌LP168菌液100ul，连续三天模型组(nos-gal4；kdm5 RNAi35706)：羽化三天的卵巢生殖系KDM5基因下调果蝇模型治疗组(nos-gal4；kdm5RNAi35706+LP168)：羽化三天的卵巢生殖系KDM5基因下调果蝇，每天一次喂食新鲜的，浓度为108CFU/ml的植物乳杆菌LP168菌液 100ul，连续三天
3.1.2 DAPI染色统计果蝇成熟卵子数量
新出生果蝇，正常培养3天后在磷酸盐缓冲盐水(PBS)中解剖，用4％多聚甲醛固定，PBS溶液清洗三遍后，用防荧光萃灭剂封片。最后用共聚焦显微镜成像计数,每组做3个生物学重复，每个重复统计20对卵巢。实验数据以平均值士标准误(SE)表示，采用T检验进行显著性分析，P<0.05为差异显著，P＜0.01为差异极显著。
3.1.3孵化率
将1日龄雄蝇与4日龄达到产卵高峰期的处女蝇放在一起使其自由交配， 12h后去除雄蝇，留下所有雌蝇产卵。用葡萄汁-琼脂-蔗糖配成的收集盛收集雌蝇产的卵，当一个收集盘上产的卵数达到约200粒时，更换新的收集盘。将带有卵的收集盛在25℃下孵化36h之后，统计孵化的卵数和总卵数的比值，即为卵孵化率。解剖镜下可观察到已孵化的卵只剩下干瘪的卵壳，未孵化的卵呈飽满的米粒状。每种交配组做3个生物学重复，实验数据以平均值士标准误(SE)表示，采用T检验进行显著性分析，P<0.05为差异显著，P＜0.01为差异极显著。
3.2实验结果
在以上四组计数每对卵巢成熟卵子的数量并进行分析。在模型组 (nos-gal4；kdm5 RNAi35706)中，相对于对照组(nos-gal4；GFP RNAi)，果蝇卵巢成熟卵子数量显著减少，通过植物乳杆菌L168治疗后(nos-gal4；kdm5 RNAi35706+LP168)，卵巢中成熟卵子数量得到提高并恢复至正常水平。
在以上四组统计产卵量及孵化率。模型组(nos-gal4；kdm5 RNAi35706)雌性果蝇产卵量显著性降低，并且子代胚胎孵化率较对照组(nos-gal4；GFP RNAi)，下降，通过植物乳杆菌L168治疗后，果蝇产卵量得到提高，并且胚胎孵化率恢复至正常水平。(设置三个实验重复，t检验，p<0.05,**p<0.01,***p<0.001)
实施例4植物乳杆菌L168提高衰老果蝇导致果蝇成熟卵子数量及子代孵化率
4.1分组信息
年轻组(WT 10day)：羽化10天的年轻野生型果蝇
年轻治疗组(WT 10day+LP168)：羽化10天的年轻野生型果蝇，每天一次喂食新鲜的，浓度为108CFU/ml的植物乳杆菌LP168菌液100ul，连续三天
衰老组(WT 50day)：羽化50天的衰老野生型果蝇
衰老治疗组(WT 50day+LP168):羽化50天的衰老野生型果蝇，每天一次喂食新鲜的，浓度为108CFU/ml的植物乳杆菌LP168菌液100ul，连续三天
按照实施例3的方法统计果蝇成熟卵子数量及子代孵化率.
4.2实验结果
在以上四组计数每对卵巢成熟卵子的数量并进行分析。在衰老组(WT 50day) 中，相对于年轻组(WT 10day)，果蝇卵巢成熟卵子数量显著减少，通过植物乳杆菌L168治疗后(WT 50day+LP168)，果蝇卵巢中成熟卵子数量得到提高并恢复至正常水平。(卵巢数量为20对，设置三个实验重复，t检验，p<0.05,**p<0.01,***p<0.001)
统计产卵量及孵化率，衰老组(WT 50day)雌性果蝇产卵量较年轻组(WT 10day)显著性降低，并且胚胎孵化率较年轻组(WT 10day)下降，通过植物乳杆菌L168 治疗后，果蝇产卵量得到提高，并且子代胚胎孵化率恢复至正常水平。
(设置三个实验重复，t检验，p<0.05,**p<0.01,***p<0.001)
实施例5植物乳杆菌L168改善了kdm5基因缺失导致卵母细胞提前激活的现象
5.1实验方法
EU转录水平监测：参照试剂盒说明书(Click-iT RNA Alexa Fluor 594ImagingKit；ThermoFisher Scientific)
5.2分组信息
对照组(nos-gal4；GFP RNAi)：羽化3天的野生型果蝇
对照治疗组组(nos-gal4；GFP RNAi+LP168)：刚羽化的新生野生型果蝇，每天一次喂食新鲜的，浓度为108CFU/ml的植物乳杆菌LP168菌液100ul，连续三天。模型组(nos-gal4；kdm5 RNAi35706)：羽化三天的卵巢生殖系KDM5基因下调果蝇模型治疗组(nos-gal4；kdm5 RNAi35706+LP168)：羽化三天的卵巢生殖系KDM5基因下调果蝇，每天一次喂食新鲜的，浓度为108CFU/ml的植物乳杆菌LP168菌液100ul，连续三天。
5.3实验结果
在模型组中(nos-gal4；kdm5 RNAi35706)，相对于对照组(nos-gal4；GFP RNAi)，卵母细胞在减数分裂阻滞期间发生了转录提前激活的现象，在模型组治疗组(nos-gal4；kdm5RNAi35706+LP168)中，LP168阻止了卵母细胞在减数分裂阻滞期间对转录提前激活。(设置三个实验重复，每个重复设置20个样本，t检验，p<0.05,**p<0.01,***p<0.001)
实施例6植物乳杆菌L168改善了自然衰老果蝇的滋养细胞提前激活的现象
6.1分组信息
年轻组(WT 10day)：羽化10天的年轻野生型果蝇
年轻治疗组(WT 10day+LP168)：羽化10天的年轻野生型果蝇，每天一次喂食新鲜的，浓度为108CFU/ml的植物乳杆菌LP168菌液100ul，连续三天。
衰老组(WT 50day)：羽化50天的衰老野生型果蝇
衰老治疗组(WT 50day+LP168):羽化50天的衰老野生型果蝇，每天一次喂食新鲜的，浓度为108CFU/ml的植物乳杆菌LP168菌液100ul，连续三天
6.2实验结果
在衰老组(WT 50day)中，相对于对照组(WT 10day)滋养细胞在减数分裂阻滞期间发生了转录提前激活的现象，在衰老治疗组(WT 50day+LP168)中，LP168 阻止了滋养细胞在减数分裂阻滞期间对转录提前激活。(设置三个实验重复，每个重复设置20个样本，t检验，p<0.05,**p<0.01,***p<0.001)。
Claims (1)
1.植物乳杆菌L168在制备延缓卵巢衰老的药物中的应用，所述的植物乳杆菌L168于2017年2月13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CGMCC，保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMC CNo.13660。
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