CN113975301B - 一种用于提高精子活力的益生菌制剂及其用途 - Google Patents
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Abstract
本发明属于生物医药技术领域，涉及一种用于提高精子活力的益生菌制剂，以及该益生菌制剂的用途。本发明所述的用于提高精子活力的益生菌制剂，包括小克里斯滕森氏菌(Christensenellaminuta)。本发明提供了所述的益生菌制剂用于制备提高精子活力的保健食品的用途，还提供了所述的益生菌制剂用于制备提高精子活力的药物的用途。本发明为保护雄性生殖健康提供新的产品，为提高雄性精子质量提供可靠的理论依据，具有重要意义，在益生菌类保健食品市场上具有良好的应用场景。
Description
技术领域
本发明属于生物医药技术领域，涉及一种用于提高精子活力的益生菌制剂，以及该益生菌制剂的用途。
背景技术
男性生育力问题日渐突出，不孕不育和生育力受损已成为全球性的健康问题。由于精子质量下降，因男性因素而导致的不育占不孕不育的40％～50％，已成为一个亟待解决的难题。统计表明，很大比例的年轻男性精子数量低于生育阈值。研究表明，精子质量的下降与疾病、药物服用、化疗、环境毒物、高龄、肥胖、西方化饮食和抽烟酗酒等多种因素相关。随着现代化进程的加快，影响生育力的因素越发普遍。在不孕不育率居高不下及“三胎”政策的大背景下，生育力的提高尤为迫切，药物治疗、辅助生殖技术、改善生活方式及膳食干预等方式是常用的治疗方式。其中，处方药和辅助生殖技术等临床治疗方式，效果虽佳但存在药物副作用强、费用昂贵、风险大等缺点。生活方式调整和膳食干预具有无创安全方便，在日常生活和临床治疗均适用等优点，但其治疗时间长，需要个性化定制。
肠道菌群对宿主健康非常重要，大量研究表明肠菌在肠道炎症机理中扮演重要的角色。近年来在膳食干预、营养保健领域，益生菌制剂的市场接受率、占比率逐年提升，来自ZION Market Research的数据显示，预计到2025年，全球益生菌产业产值将超过770亿美元，中国市场占比将超过25％。
益生菌是可定殖在人体内，并改善宿主健康的活微生物。通过膳食补充，这类微生物大多定居于人体肠道，以起到调节肠道菌群，促进营养消化吸收、改善身体机能等积极作用。而肠道微生物组被称为人体第二大基因组，对身体健康有着巨大影响。许多疾病的发生，肠道微生物都是促进因素，譬如炎症性肠病、抑郁、代谢综合征及心脑血管疾病等等。近来研究发现，男性生殖健康与肠道菌群密切相关。研究表明，高脂肪饮食诱导的肠道微生物失调会导致男性不育，这表明肠道微生物与精子发生之间存在因果关系。科学证据表明，膳食补充益生菌可一定程度减轻精子损伤。哮喘患者服用益生菌制剂，提高了自身精子质量。但关于益生菌补充剂对男性生育潜力影响的研究仍然较少。
克里斯滕森菌科(Christensenellaceae)属于厚壁菌门，在人类和动物肠道黏膜中广泛存在。目前发现的克里斯滕森菌科菌种有Christensenellaminuta(C.minuta)。小克里斯滕森氏菌(Christensenellaminuta)最初从一名健康日本男性的粪便中培育分离所得，并于2012年首次被确切描述为克里斯滕森科中的一个种。基于16S rRNA基因序列分析，将其命名为Christensenellaminuta DSM 22607(Morotomi,Masami,Fumiko Nagai,andYohei Watanabe."Description of Christensenellaminuta gen.nov.,sp.nov.,isolated from human faeces,which forms a distinct branch in the orderClostridiales,and proposal of Christensenellaceae fam.nov."Internationaljournal of systematic and evolutionary microbiology 62.1(2012):144-149.)。2014年，发现C.minuta有高度遗传性，是人类最易遗传的一类细菌(Goodrich,Julia K.,etal."Human genetics shape the gut microbiome."Cell 159.4(2014):789-799.)。此后在不同人群和多项研究中，发现人体内克里斯滕森科相对丰度与身体质量指数(Body MassIndex,BMI)呈负相关(Waters,Jillian L.,and Ruth E.Ley."The human gut bacteriaChristensenellaceae are widespread,heritable,and associated with health."BMCbiology 17.1(2019):1-11.)。
目前大多研究发现，C.minuta丰度降低甚至缺失，与肥胖和炎症性肠病如克罗恩病、溃疡性结肠炎和肠易激综合征有潜在的关系。然而结果均源于疾病状态下C.minuta在16s或宏基因组测序水平的变化，缺少机制探讨和相关有力的证据。
发明内容
本发明的第一个目的在于提供一种用于提高精子活力的益生菌制剂。
本发明所述的用于提高精子活力的益生菌制剂，包括小克里斯滕森氏菌(Christensenellaminuta)。
根据本发明所述的益生菌制剂的进一步特征，所述提高精子活力包括：精子形态完整性提高、精子畸形率降低、精子活率提高、精子头侧摆幅度提高、精子的线性度提高、鞭打频率提高、平均角位移增加、前向性增加、直线运动速率增加、平均路径速率增加、曲线速率增加中的一种或一种以上的组合。
本发明的第二个目的在于提供所述的益生菌制剂用于制备提高精子活力的保健食品的用途。
本发明的第三个目的在于提供所述的益生菌制剂用于制备提高精子活力的药物的用途。所述药物可制备成负载小克里斯滕森氏菌(Christensenellaminuta)的任何形式的制剂，如片剂、胶囊、颗粒等。
根据本发明所述的用途的进一步特征，所述提高精子活力包括：精子形态完整性提高、精子畸形率降低、精子活率提高、精子头侧摆幅度提高、精子的线性度提高、鞭打频率提高、平均角位移增加、前向性增加、直线运动速率增加、平均路径速率增加、曲线速率增加中的一种或一种以上的组合。
本发明建立了评价小克里斯滕森氏菌干预对雄性生殖能力的动物模型，发现小克里斯滕森氏菌可以作为提高雄性精子活力的保护成分。
具体地，利用计算机辅助精液分析系统(Computer-aided Semen AnalysisSystem，CASA)对精子分别进行连续帧动态和单帧静态分析，探讨小克里斯滕森氏菌(Christensenellaminuta)对精子形态、精子活力及运动相关参数的影响，评价小克里斯滕森氏菌(Christensenellaminuta)对雄性精子活力、雄性生殖能力的互作关系。
研究发现，小克里斯滕森氏菌(Christensenellaminuta)可有效提高雄性生殖能力。小克里斯滕森氏菌(Christensenellaminuta)干预可显著的降低精子畸形率，明显提高精子活率、活力及相关运动参数水平，以提高精子质量，进而提高生殖能力。
综上所述，小克里斯滕森氏菌(Christensenellaminuta)可有效提高雄性生殖能力。小克里斯滕森氏菌(Christensenellaminuta)干预显著提高了精子形态完整性、降低了精子畸形率、明显提高精子活率、活力及相关运动参数水平，以提高精子质量。本发明为未来小克里斯滕森氏菌(Christensenellaminuta)作为益生菌或相关辅助药物提高生殖能力，改善因疾病诱导的男性生殖损伤提供可靠的理论依据，具有重要意义。
附图说明
图1为小克里斯滕森氏菌(Christensenellaminuta)干预对精子形态和畸形率的影响。(A)精子畸形率(％)；(B)对照组和(C)小克里斯滕森氏菌干预后的精子形态。黄色箭头指示具有异常形态的精子，P<0.05表示组间差异性显著，P<0.1表示组间存在统计性差异。
图2为小克里斯滕森氏菌(Christensenellaminuta)干预对精子活力、活力率和相关运动参数水平的影响。(A)精子活力；(B)精子活率；(C)精子头侧摆幅度(Amplitude oflateral head displacement，ALH)；(D)精子的线性度(Linearity，LIN)；(E)鞭打频率(Beat cross frequency，BCF)；(F)平均角位移(Mean angular displacement，MAD)。P<0.05表示组间差异性显著，P<0.1表示组间存在统计性差异。
图3为小克里斯滕森氏菌(Christensenellaminuta)干预对精子相关运动参数水平的影响。(A)前向性(Straightness coefficient，STR；(B)直线运动速率(Straight-linevelocity，VSL)；(C)平均路径速率(Average path velocity，VAP)；(D)曲线速率(Curvilinear velocity，VCL)。P<0.05表示组间差异性显著，P<0.1表示组间存在统计性差异。
具体实施方式
下面结合说明书附图及具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1小克里斯滕森氏菌(Christensenellaminuta)对干预对精子质量的影响。
1.实验材料：小克里斯滕森氏菌(Christensenellaminuta,DSM22607)来自德国微生物菌种保藏中心(DSMZ)。
2.实验动物：C57BL/6JNifdc雄性小鼠，购自浙江维通利华实验动物技术有限公司(合格证编号：20210526Abzz0619000720)。在环境为光照时间昼夜12h交替，温度为20-25℃，相对湿度为50±5％的暨南大学实验动物中心适应性饲养，自由摄食。实验操作均按照《暨南大学实验动物伦理委员会》相关要求进行。
3.实验方法
(1)实验动物分组及模型建立
采用普通饲料对C57BL/6JNifdc雄性小鼠进行适应性喂养，利用体重分层法对16只小鼠随机平均分2组，每组8只，分别为：Conrol组和C.minuta组，给予普通饮用水自由饮用9天。
(2)干预处理
所有组别小鼠均在第一天开始进行干预，且干预至实验结束：其中Control组灌胃相应体积的水，而C.minuta组灌胃相应体积的小克里斯滕森氏菌(Christensenellaminuta)干预物。
(3)精子参数测定
a.提前半小时开启精子分析仪，并对精子计数板进行37℃预热。同时，提前半小时开启水浴锅对生理盐水进行37℃预热。
b.将右侧附睾尾置于1.0mL事先预热好生理盐水中迅速剪碎，37℃释放5分钟后，，取10.0μL充分混匀了的精子混悬液加入精子计数板中，利用计算机辅助精液分析系统(Computer-aided Semen Analysis System，CASA)对精子进行连续帧动态拍照，用于测定精子活力及运动相关参数。随后待精子静止不动后，对精子进行单帧静态拍照，用于精子数量及形态分析。
c.根据拍摄图片，结合CASA系统对精子进行动态和静态分析。
4.实验结果
本发明成功构建了评价小克里斯滕森氏菌(Christensenellaminuta)干预对雄性生殖能力影响的模型。采用计算机辅助精液分析系统，对精子形态及功能进行深入的探讨。与Control组相比，小克里斯滕森氏菌(Christensenellaminuta)处理组出现显著提高精子质量的表型：精子形态完整性提高，精子畸形率降低，精子活力和精子活率等精子相关运动参数水平均明显提高，直线运动速率、平均路径速率和曲线速率均明显提高(图2-图3)。本发明指出了小克里斯滕森氏菌(Christensenellaminuta)在提高精子质量的作用，发现小克里斯滕森氏菌(Christensenellaminuta)可以作为提高雄性精子活力和改善雄性生殖能力的保护成分。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，对于本领域的普通技术人员来说，在上述说明及思路的基础上还可以做出其它不同形式的变化或变动，这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。
Claims (2)
1.包括小克里斯滕森氏菌（Christensenella minuta）的益生菌制剂用于制备提高精子活力的药物的用途，所述小克里斯滕森氏菌是来自德国微生物菌种保藏中心，保藏编号为DSM22607。
2.根据权利要求1所述的用途，其特征在于，所述提高精子活力包括：精子形态完整性提高、精子畸形率降低、精子活率提高、精子头侧摆幅度提高、精子的线性度提高、鞭打频率提高、平均角位移增加、前向性增加、直线运动速率增加、平均路径速率增加、曲线速率增加中的一种或一种以上的组合。
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