CN117551601A - 8-异戊烯基黄酮在改善衰老卵母细胞质量和整倍性中的用途 - Google Patents
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Abstract
本发明属于生物医药领域，公开了8‑异戊烯基黄酮在改善衰老卵母细胞质量和整倍性中的用途，所述用途不包括疾病的诊断和治疗；进一步的，本发明具体公开了8‑异戊烯基黄酮在制备改善衰老卵母细胞质量和整倍性的试剂/药物中的用途，包括在卵丘‑卵母细胞复合物体外成熟培养基中添加8‑异戊烯基黄酮，可以大幅改善衰老卵母细胞减数分裂进程和成熟卵母细胞质量；同时发现，体内给药也可改善衰老小鼠卵巢储备和卵母细胞质量；因此我们发现，8‑异戊烯基黄酮在制备卵丘‑卵母细胞复合物(COCs)体外成熟培养基以及改善衰老卵母细胞质量和整倍性的药物中具有良好的应用前景。
Description
技术领域
本发明属于生物医药领域，公开了8-异戊烯基黄酮在改善衰老卵母细胞质量和整倍性中的用途。
背景技术
卵巢衰老是以卵巢功能进行性下降为特征，表现为卵母细胞数量和质量随年龄的增长而下降。与其他器官相比，卵巢是最早显示衰老特征的器官系统之一。大多数国家,首次怀孕的高生育年龄(≥35岁)妇女日益增多。随着年龄的增长，受孕困难和不孕症增加；高龄妇女的卵母细胞有更高的机会导致流产和/或后代出生缺陷。在整个减数分裂进程中，衰老的卵母细胞更容易出现姐妹染色单体的反向分离、过早分离、不分离或染色体滞后等现象，导致高龄妇女的卵母细胞非整倍体率显著增高。前期研究表明，除了运用干细胞治疗卵巢损伤，还包括如线粒体治疗、抗氧化剂、表观遗传调节剂、端粒酶激活剂和中药等已被用于预防卵巢衰老，而这些疗法中的大多数尚未进行临床试验或临床干预效果不显著。卵巢代谢微环境、颗粒细胞和卵母细胞代谢偶联改变是机体代谢影响卵母细胞质量和减数分裂进程的重要原因。
8-异戊烯基黄酮是8-异戊烯基黄酮苷类化合物之一。它具有异戊烯基侧链，从狭义上讲，异戊烯基侧链是指含有5C的异戊烯基侧链，广义上还包括10C的香叶基侧链和15C的法尼基侧链。研究发现，异戊烯基侧链增加了黄酮的亲脂性，改善了其对生物膜P-糖蛋白的亲和力，从而降低了黄酮类化合物在生物细胞中的清除率。研究证实，8-异戊烯基黄酮作用于体外培养大鼠卵巢颗粒细胞可以促进颗粒细胞增殖,并增加CYP17、CYP19基因表达,从而促进颗粒细胞分泌E2和孕酮。8-异戊烯基黄酮通过以浓度和时间依赖性方式促进人卵巢颗粒样胞中的雌激素生物合成，提高芳香化酶表达，诱导卵巢颗粒细胞的增殖，增加雌激素和孕酮的产生。8-异戊烯基黄酮增加卵巢、子宫和垂体腺的重量，改善雌性大鼠发育，延长了大鼠动情周期的持续时间。但是其作为异戊烯基的供体是否可以改善衰老小鼠的卵母细胞减数分裂进程，仍不清楚。
发明内容
为解决上述问题，本发明公开了8-异戊烯基黄酮在改善衰老卵母细胞质量和整倍性中的用途，本发明发现在体外培养的卵母细胞-卵丘细胞复合体的成熟培养基中添加异戊二烯基供体8-异戊烯基黄酮，或对衰老雌性小鼠灌胃8-异戊烯基黄酮，能够改善卵巢功能，提高卵母细胞质量和减数分裂正常率。
本发明包括以下技术方案：
一方面本发明提供了8-异戊烯基黄酮在改善衰老卵母细胞质量和整倍性中的用途，所述用途不包括疾病的诊断和治疗。
进一步的，上述用途包括8-异戊烯基黄酮在制备改善衰老卵母细胞质量和整倍性的试剂/药物中的用途。
进一步的，上述用途包括在卵丘-卵母细胞复合物体外成熟培养基中添加8-异戊烯基黄酮。
进一步的，上述用途中，所述体外成熟培养基中8-异戊烯基黄酮的终浓度为40-60μg/L。
另一方面，本发明公开了一种卵丘-卵母细胞复合物体外成熟培养基，其特征在于，含有终浓度40-60μg/L的8-异戊烯基黄酮。
进一步的，上述一种卵丘-卵母细胞复合物体外成熟培养基，含有以下组分：BSA、抗生素、丙酮酸钠、谷氨酰胺、M199和8-异戊烯基黄酮。
进一步的，基于卵丘-卵母细胞复合物体外成熟培养基中的8-异戊烯基黄酮成分，制备8-异戊烯基黄酮口服制剂。
进一步的，制备的8-异戊烯基黄酮口服制剂，所述试剂/药物的剂量范围为4-6mg/kg体重的8-异戊烯基黄酮。
另一方面，本发明公开了一种口服的改善衰老卵母细胞质量和整倍性的制剂/药物，含有8-异戊烯基黄酮。
进一步的，获取的卵母细胞来自小鼠。
本发明具有以下有益效果：
本发明首次公开了8-异戊烯基黄酮在改善衰老卵母细胞质量和整倍性中的用途，在实施例中我们通过CCK-8实验进行浓度验证表明50μg/L是最适浓度，体外培养基中添加40-60μg/L 8-异戊烯基黄酮，通过评估COCs来源的卵母细胞减数分裂进程和非整倍体率，明确8-异戊烯基黄酮通过颗粒细胞改善衰老卵母细胞质量。为了进一步确定8-异戊烯基黄酮的疗效，在另一些实施例中，9月半龄雌鼠灌胃5mg/kg体重的8-异戊烯基黄酮，连续灌胃14天后可以显著增加卵巢重量、卵巢指数和各级卵泡的数量。8-异戊烯基黄酮给药后明显增加雌鼠COCs来源卵母细胞PB1排放率和排卵数量，显著降低MII卵母细胞染色体/纺锤体异常率，增加卵母细胞整倍性。
因此，本发明公开的8-异戊烯基黄酮在制备卵丘-卵母细胞复合物体外成熟培养基或改善衰老卵母细胞质量和整倍性的药物中具有良好的前景。
附图说明
图1为8-异戊烯基黄酮的化学结构式；
图2为8-异戊烯基黄酮体外添加至成熟培养基后显著降低卵母细胞减数分裂异常图；其中a：8-异戊烯基黄酮对小鼠卵巢颗粒细胞增殖的影响；b-c：8-异戊烯基黄酮体外添加改善衰老小鼠卵母细胞PB1排放率，标尺长度为100μm；d-e：8-异戊烯基黄酮体外添加显著降低衰老小鼠卵母细胞非整倍体率；
图3为8-异戊烯基黄酮体内给药显著改善衰老雌鼠的卵巢功能图；a-d:8-异戊烯基黄酮体内给药改善衰老小鼠卵巢功能，标尺长度为1mm，200μm，20μm；
图4为8-异戊烯基黄酮体内给药降低卵母细胞减数分裂异常图；a-b：8-异戊烯基黄酮体内给药改善衰老小鼠卵母细胞PB1排放率，标尺长度为100μm；c-f：8-异戊烯基黄酮体内给药显著降低衰老小鼠卵母细胞减数分裂异常率，标尺长度为25μm。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
本发明实施例中使用的试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。
按照如下组分在无菌的条件下配制含有50μg/L 8-异戊烯基黄酮(源叶，CAS：489-32-7，分析标准品，HPLC≥98％)的体外成熟培养基：
实施例1
(1)10月龄C57BL/6雌鼠腹腔注射10IU PMSG，48h后处死小鼠，PBS中分离卵巢，将卵巢转移到M2培养基中，使用1ml注射器刺破窦卵泡释放COCs，口吸管转移COCs至矿物油覆盖的含有50μg/L 8-异戊烯基黄酮的体外成熟培养基，37℃，5％ CO2培养箱培养。
(2)培养14h后，使用透明质酸酶消化去除颗粒细胞，统计第一极体(PB1)排放率并拍照。
(3)将卵吸于台式液中去掉透明带，每次10个左右，放恒温台上，等待数分钟，注意观察。
(4)去掉透明带后，将卵吸于M2中养10min洗净，放恒温台上。
(5)在载玻片上加20μl铺片液，将卵吸入每张片子，约10个卵，看到卵破裂。
(6)在超净台中风干。
(7)Wash洗3次，每次10min。
(8)1％BSA封闭1h。
(9)一抗孵育4℃过夜。
(10)PBST洗4次，每次15min。
(11)二抗孵育，室温1h。
(12)PBST洗3次，每次10min。用Hochest染核。
(13)防荧光淬灭剂封片，镜检，统计非整倍体率。
结果如附图2所示，表明8-异戊烯基黄酮体外添加至成熟培养基后显著降低卵母细胞非整倍体率。
实施例2
(1)将9月半龄C57BL/6小鼠分为2组，对照组和8-异戊烯基黄酮给药组，每日10:00给药，9月半龄雌鼠灌胃5mg/kg体重8-异戊烯基黄酮，连续灌胃14天。
(2)14d后，部分雌鼠腹腔注射10IU PMSG，48h后处死小鼠，PBS中分离卵巢。
(3)将卵巢转移到M2培养基中，使用1ml注射器刺破窦卵泡释放COCs，口吸管转移COCs至矿物油覆盖的M199体外成熟培养基，37℃，5％ CO2培养箱培养。
(4)培养14h后，使用透明质酸酶消化去除颗粒细胞，统计第一极体(PB1)排放率并拍照。
(5)14d后，部分雌鼠腹腔注射10IU PMSG，48h后注射HCG，注射HCG13 h后处死小鼠，获得输卵管，M2中刺破输卵管壶腹膨大部。
(6)透明质酸酶消化扩散的COCs团，取PB1排放卵母细胞在4％多聚甲醛固定30min。
(7)口吸管转移卵母细胞至0.5％Triton X-100透膜20min。
(8)1％ BSA封闭1h。
(9)1％ BSA稀释一抗(α-tublin)，口吸管转移卵母细胞至200μL一抗液滴中，置于湿盒中4℃孵育过夜。
(10)Wash避光洗涤3次，每次5min。转移卵母细胞至Hochest液滴中，室温避光染色10min。
(11)防荧光淬灭剂封片，镜检，记录纺锤体/染色体异常率。
结果如附图4所示，8-异戊烯基黄酮体内给药降低卵母细胞减数分裂异常率。
实施例3
(1)将9月半龄C57BL/6小鼠分为2组，对照组和8-异戊烯基黄酮给药组，每日10:00给药，9月半龄雌鼠灌胃5mg/kg体重8-异戊烯基黄酮，连续灌胃14天。
(2)称量各组小鼠重量，脱臼处死小鼠后，下腹部U型切口，暴露腹腔大体脏器结构，寻找双子宫体，向上延伸寻找与输卵管相连的卵巢。体视镜下剥除卵巢周围脂肪组织，拍照。拍照后的卵巢组织置于干燥无菌纱布上，待无液体残留后使用电子分析天平称重。按以下公式计算卵巢指数：卵巢指数(ovarian index)＝卵巢重量(mg)/体重(g)×100％。并进行卵巢连续切片，HE染色后卵泡计数。
(3)14d后，部分雌鼠腹腔注射10IU PMSG，48h后处死小鼠，PBS中分离卵巢。
(4)将卵巢转移到M2培养基中，使用1ml注射器刺破窦卵泡释放COCs，口吸管转移COCs至矿物油覆盖的M199体外成熟培养基，37℃，5％ CO2培养箱培养。
(5)培养14h后，使用透明质酸酶消化去除颗粒细胞，统计第一极体(PB1)排放率并拍照。
(6)14d后，部分雌鼠腹腔注射10IU PMSG，48h后注射HCG，注射HCG13 h后处死小鼠，获得输卵管，M2中刺破输卵管壶腹膨大部。
(7)透明质酸酶消化扩散的COCs团，吸取含有PB1的卵母细胞，台式液中去除透明带，每次10个左右，放温台上，等待数分钟，注意观察。
(8)去掉透明带后，将卵吸于M2中养10min洗净，放温台上。
(9)在载玻片上加20μl铺片液，将卵吸入每张片子，约10个卵，看到卵破裂。
(10)在超净台中风干。
(11)Wash洗3次，每次10min。
(12)1％BSA封闭1h。
(13)一抗孵育4℃过夜。
(14)PBST洗4次，每次15min。
(15)二抗孵育，室温1h。
(16)PBST洗3次，每次10min。用Hochest染核。
(17)防荧光淬灭剂封片，镜检，统计非整倍体率。
结果如附图3和4所示，8-异戊烯基黄酮体内给药显著改善衰老雌鼠的卵巢功能、以及8-异戊烯基黄酮体内给药降低卵母细胞非整倍体率。
以上显示和描述了本发明的基本原理、主要特征和优点。本行业的技术人员应该了解，上述实施例不以任何形式限制本发明，凡采用等同替换或等效变换的方式所获得的技术方案，均落在本发明的保护范围内。
Claims (10)
1.8-异戊烯基黄酮在改善衰老卵母细胞质量和整倍性中的用途，所述用途不包括疾病的诊断和治疗。
2.根据权利要求1所述的用途，其特征在于，包括：8-异戊烯基黄酮在制备改善衰老卵母细胞质量和整倍性的试剂/药物中的用途。
3.根据权利要求2所述的用途，其特征在于，包括在卵丘-卵母细胞复合物体外成熟培养基中添加8-异戊烯基黄酮。
4.根据权利要求3所述的用途，其特征在于，所述体外成熟培养基中8-异戊烯基黄酮的终浓度为40-60μg/L。
5.一种卵丘-卵母细胞复合物体外成熟培养基，其特征在于，含有终浓度40-60μg/L的8-异戊烯基黄酮。
6.根据权利要求5所述的一种卵丘-卵母细胞复合物体外成熟培养基，其特征在于，含有以下组分：BSA、抗生素、丙酮酸钠、谷氨酰胺、M199和8-异戊烯基黄酮。
7.根据权利要求2所述的用途，其特征在于，所述试剂/药物为口服制剂。
8.根据权利要求7所述的用途，其特征在于，所述试剂/药物的剂量范围为4-6mg/kg体重的8-异戊烯基黄酮。
9.一种口服的改善衰老卵母细胞质量和整倍性的制剂/药物，其特征在于，含有8-异戊烯基黄酮。
10.根据权利要求2所述的用途，其特征在于，所述卵母细胞来自小鼠。
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