CN114931569B - 甲基硒代半胱氨酸在制备提高雄性生殖能力的产品中的应用 - Google Patents
甲基硒代半胱氨酸在制备提高雄性生殖能力的产品中的应用 Download PDFInfo
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Abstract
本发明提供了甲基硒代半胱氨酸在制备提高雄性生殖能力的产品中的应用,属于医药技术领域,本发明提供的甲基硒代半胱氨酸在制备提高雄性生殖能力的产品中的应用,以甲基硒代半胱氨酸这一特定形态存在的硒,作用于小鼠睾丸间质细胞后,细胞的增殖能力提高相对于其他形态的硒(硒代胱氨酸、硒代蛋氨酸、硒代半胱氨酸和亚硒酸钠)最为显著,且有效浓度最为广泛。因此,甲基硒代半胱氨酸具有应用于提高雄性生殖能力的潜力,能够用于制备相关的药物和保健食品。
Description
技术领域
本发明属于医药技术领域,尤其涉及甲基硒代半胱氨酸在制备提高雄性生殖能力的产品中的应用。
背景技术
不孕不育是21世纪影响人类健康的主要问题之一。在影响不孕不育的因素中,男性方面约占50%,而精液质量降低是造成男性不育的重要因素之一(Virtanen,H.,Jorgensen,N.,Toppari,J.,et al.Semen quality in the 21 st century.NatureReviews Urology,2017,14:120-130.)。据统计,导致男性精液质量下降的原因主要分为内在因素(遗传因素)和外在因素(环境因素),尤其是环境污染的加剧使得男性生精细胞严重损伤(Hsu,P.,Huang,W.,Yao,W.,et al.Aperm changes in men exposed topolychlorinated biphenyls and dibenzofurans.The Journal of the AmericanMedical Association,2003,289:2943-2944.)。因此,生殖健康和生殖安全的提升应致力于提高男性生殖能力的研究。
硒是人体必需的微量元素之一,研究表明,硒在整个生殖系统都有分布,且睾丸中硒含量最高(Guerriero,G.,Trocchia,S.,Abdel-Gawad,F.,et al.Roles ofreactiveoxygen species in the spermatogenesis regulation.Front Endocrinol,2014,5:56.)。缺硒可导致动物体内过氧化脂类、丙二醛以及活性氧含量升高,使得睾丸曲精细管生殖细胞发育不良、精子活力下降和精液质量异常等,最终导致雄性不育(Qazi,I.,Angel,C.,Yang,H.,et al.Role of Selenium and Selenoproteins in Male ReproductiveFunction:A Review of Past and Present Evidences.Antioxidants,2019,8:268.),而通过补硒可以增强精子活力(Ghafarizadeh,A.,Vaezi,G.,Shariatzadeh,M.,etal.Effect ofin vitro selenium supplementation on sperm quality inasthenoterato-zoospermic men.Andrologia,2018,50:12896.)。目前硒的增精作用机理主要归纳为:(1)硒被动物吸收后转运到睾丸,在一系列脂质蛋白受体的作用下合成睾丸硒蛋白,维持精子的正常生成(Noblanc,A.,Peltier,M.,Damon-Soubeyrand,C.,etal.Epididymis response partly compensates for spermatozoa oxidative defectsin snGPx4 and GPx5 double mutant mice.PLoS One,2012,7:38565.);(2)硒作为硒酶和硒蛋白(GPx4、mGPx5和snGPx4)的重要组成部分能够及时清除睾丸组织中的过氧化物,保护精细胞免受活性氧的损伤,促进睾酮和雄激素结合蛋白生成,提高精液质量(Ahsan,U.,Kamran,Z.,Raza,I.,et al.Role ofselenium in male reproduction-areview.AnimalReproduction Science,2014,146:55-62.)。
硒是一种易排泄的元素,人体每天必须摄入足够量的硒。硒的增精功能与补硒量紧密相关,适宜剂量的硒对精液有促进作用,而过量会对精子产生毒害(Stuss,M.,Michalska-Kasiczak,M.,Sewerynek,E.The role ofselenium in thyroid glandpathophysiology.EndokrynologiaPolska,2017,68:440-465.);此外,硒的增精功能也取决于硒的形态,相比无机硒,有机硒的补充使得动物精子浓度更高和质量更好(Liu,F.,Cottrell,J.,Furness,J.,et al.Selenium and vitamin E together improve intestinalepithelial barrier function and alleviate oxidative stress in heat-stressedpigs.Experimental Physiology,2016,101:801-810.)。
目前相关的机制研究工作主要集中采用无机硒(Na2SeO3),少量研究采用酵母硒和硒代蛋氨酸(SeMet)完成相关实验。因此,确定具有最佳增精作用效果的特定硒形态及其使用浓度对提高雄性生殖能力具有重要意义。
发明内容
有鉴于此,本发明的目的在于提供甲基硒代半胱氨酸在制备提高雄性生殖能力的药物中的应用,所述甲基硒代半胱氨酸具有促进生殖细胞活性的功能,能够应用于制备增精药物或增精食品。
本发明提供了甲基硒代半胱氨酸在制备提高雄性生殖能力的药物中的应用。
本发明还提供了甲基硒代半胱氨酸在制备提高睾丸间质细胞的增殖能力的药物中的应用。
优选的,所述睾丸间质细胞包括小鼠睾丸间质细胞。
优选的,所述甲基硒代半胱氨酸的有效浓度为1000~10000μg/L。
优选的,所述甲基硒代半胱氨酸的有效浓度为2000~6000μg/L。
优选的,所述甲基硒代半胱氨酸的有效浓度为3000~5000μg/L。
本发明还提供了甲基硒代半胱氨酸在制备保健食品中的应用。
本发明还提供了甲基硒代半胱氨酸在睾丸间质细胞增殖培养中的应用,将甲基硒代半胱氨酸与对数生长期的睾丸间质细胞混合后培养。
优选的,所述睾丸间质细胞的细胞浓度为(0.5~1.5)×105cell/mL。
优选的,所述甲基硒代半胱氨酸在睾丸间质细胞培养体系中的终浓度为1000~10000μg/L。
与现有技术相比,本发明具有如下有益效果:本发明提供的甲基硒代半胱氨酸在制备提高雄性生殖能力的产品中的应用,以甲基硒代半胱氨酸这一特定形态存在的硒,作用于小鼠睾丸间质细胞后,细胞的增殖能力提高相对于其他形态的硒(硒代胱氨酸、硒代蛋氨酸、硒代半胱氨酸和亚硒酸钠)最为显著,且有效浓度最为广泛。因此,甲基硒代半胱氨酸具有应用于提高雄性生殖能力的潜力,能够用于制备相关的药物和保健食品。
进一步的,鉴于甲基硒代半胱氨酸能够提高睾丸间质细胞的增殖活性,能够应用于培养睾丸间质细胞以获得更高活性和浓度的睾丸间质细胞,用于科学实验研究。
附图说明
图1为不同浓度甲基硒代半胱氨酸对小鼠睾丸间质细胞存活率的影响图(孵育时间分别为12、24和48h;细胞密度为100000cells/mL);
图2为不同浓度硒代半胱氨酸对小鼠睾丸间质细胞存活率的影响图(孵育时间分别为12、24和48h;细胞密度为100000cells/mL);
图3为不同浓度硒代胱氨酸对小鼠睾丸间质细胞存活率的影响图(孵育时间分别为12、24和48h;细胞密度为100000cells/mL);
图4为不同浓度硒代蛋氨酸对小鼠睾丸间质细胞存活率的影响图(孵育时间分别为12、24和48h;细胞密度为100000cells/mL);
图5为不同浓度亚硒酸钠对小鼠睾丸间质细胞存活率的影响图(孵育时间分别为12、24和48h;细胞密度为100000cells/mL)。
具体实施方式
本发明提供了甲基硒代半胱氨酸在制备提高雄性生殖能力的药物中的应用。在本发明中,以甲基硒代半胱氨酸这一特定形态存在的硒能够显著提高睾丸间质细胞的增殖能力,高睾丸间质细胞的增殖能力对于增强精子活力有影响,因此,甲基硒代半胱氨酸能够用于制备提高雄性生殖能力的药物。
本发明还提供了甲基硒代半胱氨酸在制备提高睾丸间质细胞的增殖能力的药物中的应用。
在本发明中,所述睾丸间质细胞包括但不限于小鼠睾丸间质细胞。
在本发明中,所述甲基硒代半胱氨酸的有效浓度优选为1000~10000μg/L,进一步有优选为2000~6000μg/L,更进一步优选为3000~5000μg/L。
本发明提供的甲基硒代半胱氨酸在上述限定的较为宽泛的浓度范围内均具有显著的提高睾丸间质细胞活性的作用,甲基硒代半胱氨酸这一特定形态,效果显著,且有效浓度范围广,更适于应用。
本发明对上述药物的剂型和辅料没有特殊限定,采用甲基硒代半胱氨酸药学上可接受的辅料和剂型即可。
本发明还提供了甲基硒代半胱氨酸在制备保健食品中的应用。本发明对所述保健食品的剂型以及辅料没有特殊限定,采用本领域常规的保健食品剂型和辅料即可。
本发明还提供了甲基硒代半胱氨酸在睾丸间质细胞增殖培养中的应用,将甲基硒代半胱氨酸与对数生长期的睾丸间质细胞混合后培养。
本发明鉴于甲基硒代半胱氨酸能够提高睾丸间质细胞的增殖活性的功效,将其应用于培养睾丸间质细胞以获得更高活性和浓度的睾丸间质细胞,为科学实验研究提供细胞材料。
在本发明中,所述睾丸间质细胞的细胞浓度优选为(0.5~1.5)×105cell/mL,进一步优选为(0.8~1.2)×105cell/mL,更进一步优选为1×105cell/mL。
在本发明中,所述甲基硒代半胱氨酸在睾丸间质细胞培养体系中的终浓度为1000~10000μg/L,进一步有优选为2000~6000μg/L,更进一步优选为3000~5000μg/L。在本发明中,所述睾丸间质细胞优选为小鼠睾丸间质细胞。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例中涉及的Se-甲基硒代L-半胱氨酸(MeSeCys)的分子式为C4H9NO2Se;L-硒代胱氨酸(SeCys2)的分子式为C6H12N2O4Se2;硒代蛋氨酸(SeMet)的分子式为C5H11NO2Se;硒代L-半胱氨酸(SeCys)的分子式为C3H7NO2Se。
实施例1
甲基硒代半胱氨酸体外促进小鼠睾丸间质细胞活性研究
小鼠睾丸间质细胞由油料品质化学与加工利用团队(中国农业科学院油料作物研究所,武汉,中国)提供,也可采用市售的小鼠睾丸间质细胞。用T25培养瓶培养小鼠睾丸间质细胞,观察T25培养瓶中的小鼠睾丸间质细胞是否铺满(80%以上最宜)。取对数生长期的细胞,弃掉T25培养瓶中的培养基,用PBS清洗细胞,然后迅速用1mL 0.25%的胰蛋白酶消化细胞。消化完成后,加入2mL细胞培养基终止消化。将细胞悬液于离心机中1000rpm离心3min,弃上清,加入2mL细胞培养基,将细胞混匀。利用细胞计数仪计算活细胞个数,配置成1×105cell/mL的细胞悬液,以每孔100μL加入96孔板,继续培养24h。24h后从培养箱中取出96孔板观察细胞是否已经贴壁。观察完毕后吸出每孔的培养基。
添加10~10000μg/L浓度范围(分别为10、50、1000、2000、4000、6000、8000、10000μg/L)的甲基硒代半胱氨酸溶液,每孔100μL,每一个浓度都设立6个平行孔,分别继续培养12、24和48h,吸出每孔中的培养基,将CCK-8与培养基按照1:9的体积比混匀,每孔加入100μL,37℃孵育40min。将96孔板在酶标仪内设定程序首先振荡15s,之后于450nm的波长下测定板中各孔的OD值。空白组采用培养基代替细胞样品,其他步骤操作一致。对照组中细胞样品采用不含硒的培养基培养,其他处理步骤同上。
以公式(如下)计算小鼠睾丸间质细胞的存活率:
细胞存活率(%)=(实验组OD值-空白组OD值)/(对照组OD值-空白自OD值)×100%。
结果如图1和表1所示,采用甲基硒代半胱氨酸孵育生殖细胞时,在甲基硒代半胱氨酸孵育浓度为1000~10000μg/L时,细胞增殖率明显增加,最大效应值可达到56.58%。
表1不同浓度甲基硒代半胱氨酸对小鼠睾丸间质细胞存活率(%)的影响
对比例1
硒代半胱氨酸体外促进小鼠睾丸间质细胞活性研究
对比例1与实施例1的区别仅在于,细胞孵育处理:用10~10000μg/L(分别为10、50、1000、2000、4000、6000、8000、10000μg/L)浓度范围的硒代半胱氨酸溶液分别处理小鼠睾丸间质细胞12、24和48h。
结果如图2所示,在硒代半胱氨酸孵育浓度为10~10000μg/L范围内时,硒代半胱氨酸对细胞无明显的促增殖作用。
对比例2
硒代胱氨酸体外促进小鼠睾丸间质细胞活性研究
对比例2与实施例1的区别仅在于,细胞孵育处理:用10~10000μg/L(分别为10、50、200、500、1000、2000、3000、5000μg/L)浓度范围的硒代胱氨酸溶液分别处理小鼠睾丸间质细胞12、24和48h。
结果如图3所示,仅在采用1000μg/L硒代胱氨酸孵育小鼠睾丸间质细胞24h时,细胞增殖率有所升高;在其它孵育浓度条件下,硒代胱氨酸对细胞无明显的促增殖作用,且随着孵育浓度的增加,细胞增殖率有所降低。
对比例3
硒代蛋氨酸体外促进小鼠睾丸间质细胞活性研究
对比例3与实施例1的区别仅在于,细胞孵育处理:用10~10000μg/L(分别为10、50、1000、2000、4000、6000、8000、10000μg/L)浓度范围的硒代蛋氨酸溶液分别处理小鼠睾丸间质细胞12、24和48h。
结果如图4所示,在硒代蛋氨酸孵育浓度为10~10000μg/L范围内时,硒代蛋氨酸对细胞无明显的促增殖作用。
对比例4
亚硒酸钠体外促进小鼠睾丸间质细胞活性研究
对比例4与实施例1的区别仅在于,细胞孵育处理:用10~5000μg/L(分别为10、50、200、500、1000、2000、3000、5000μg/L)浓度范围的亚硒酸钠溶液分别处理小鼠睾丸间质细胞12、24和48h。
结果如图5所示,在亚硒酸钠孵育浓度为10~5000μg/L范围内时,亚硒酸钠对细胞无明显的促增殖作用,且随着孵育浓度的增加,细胞增殖率有所降低。
对比Na2SeO3、SeCys2、MeSeCys、SeCys和SeMet孵育小鼠睾丸间质细胞后细胞存活率的变化(图1至图5),从有效浓度和最大效应值两方面可以看出,MeSeCys促进小鼠睾丸间质细胞活性的作用最为显著。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.甲基硒代半胱氨酸在制备提高睾丸间质细胞的增殖能力的药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述睾丸间质细胞为小鼠睾丸间质细胞。
3.根据权利要求1或2所述的应用,其特征在于,所述甲基硒代半胱氨酸的有效浓度为1000~10000μg/L。
4.根据权利要求3所述的应用,其特征在于,所述甲基硒代半胱氨酸的有效浓度为2000~6000μg/L。
5.根据权利要求4所述的应用,其特征在于,所述甲基硒代半胱氨酸的有效浓度为3000~5000μg/L。
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