CN111643442A - 一种外泌体美容液及其制备方法 - Google Patents
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Abstract
本发明提供一种外泌体美容液及其制备方法，涉及生物细胞护肤品技术领域，包括相应配比的脐带间充质干细胞外泌体溶液、甘草外泌体、香菇多糖、生育酚、透明质酸钠的混合物，通过使用微针导入，达到改善面部细纹、提亮肤色的功效。包括以下步骤：使用获赠的健康母体的脐带制备出间充质干细胞，通过超高速离心获得脐带间充质干细胞外泌体溶液；通过差速离心等技术获得甘草外泌体，将脐带间充质干细胞外泌体溶液、甘草外泌体、香菇多糖、生育酚、透明质酸钠按照一定比例混合，制备出具有提高面部代谢能力、改善面部细纹、提亮肤色功效的外泌体美容液。
Description
技术领域
本发明涉及生物细胞护肤品的技术领域，尤其涉及一种外泌体美容液及其制备方法。
背景技术
干细胞是一种未分化的多能细胞，可以分化成具有特异功能的特化细胞。干细胞疗法成为治疗多种疾病的首选方法。干细胞一词最早出现在1868年由Ernst Haeckel撰写的科学文献中。然而，直到20世纪60年代才发现造血***中存在干细胞的科学依据，科学家在其中发现了克隆性骨髓前体，并将其注射到受辐射的小鼠体内后，在小鼠脾脏部位产生了大量的集落。
在此基础上，科学家发现了干细胞的特征：具有长期自我更新和多向分化的能力。干细胞在再生医学上的应用也非常广泛，例如改善皮肤皱纹、均匀肤色、提高免疫力、增强代谢功能等，其商业价值巨大。
间充质干细胞(MSCs)是广泛分布的细胞，具有自我更新和多向分化的能力。间充质干细胞目前已在开展用于恶性白血病、移植物抗宿主病、急性心梗等疾病。在再生医学上，间充质干细胞用于改善亚健康状态、延缓衰老。脐带间充质干细胞数量多，增殖能力强，具有基因稳定、不易突变、免疫原性低，使用时无需配型。
外泌体是由细胞分泌的直径为30-150nm的微细胞囊泡，具有脂质双层膜结构。它包含成千上万种细胞特异性蛋白质、脂质和核酸，并且在许多生理和病理过程中发挥重要作用，例如免疫力中的抗原呈递，组织损伤的修复等，并且是细胞免疫的通讯载体，也是细胞***和再生的重要物质。间充质干细胞可较其他细胞种类（如成肌细胞、胚肾细胞等）分泌出更多的外泌体。
人一般在25岁时开始衰老，并且在快速发展的社会，环境、饮食、工作压力等因素会加快衰老速度，多数人为亚健康、早衰状态，表现在神经***方面，如失眠、易疲劳、精力不足、记忆力减退等，在生殖***方面，如性功能下降、卵巢早衰、早更综合症等，在皮肤***方面，如皮肤粗糙、色斑、皱纹产生、皮肤松弛等，在运动***方面，如肌肉乏力，腰膝酸软疼痛、骨质疏松等，在免疫***方面，如免疫力下降、易感染疾病等，在代谢***方面，如新陈代谢速度减慢、出现肥胖等一系列问题。市面上针对于亚健康及早衰有一系列的保健品及护肤品，但是效果不佳，或会反弹，因此开发一个效果持续且健康的抗衰产品是一个急需解决的问。
发明内容
本发明的目的是提供一种外泌体美容液及其制备方法，以解决现有技术问题，实现提高面部代谢能力、改善面部细纹、提亮肤色等功效。
本发明的目的是这样实现的：
脐带间充质干细胞外泌体的制备：
将脐带从脐带装载瓶中取出，用生理盐水洗涤，并剪成1.5-2厘米的段状，继续用生理盐水洗涤2-3次，取出华氏胶，洗涤后剪碎呈米粒大小，平铺于培养皿，倒置15-30分钟，加入人间充质干细胞培养基和添加剂开始培养，3天后换液，之后每隔2-3天换液一次，至细胞达到80-85%融合结束原代培养，用0.25%胰酶消化，镜下观察细胞呈圆形，加入同体积的胎牛血清终止消化，用PBS洗涤后加入人间充质干细胞培养基重悬，按1:2传代，分瓶培养，取第2-3代细胞生存状态比较好的细胞，用无血清培养基培养48h，收集培养上清液，除去细胞碎片，采用100-280分钟离心力为200000g的超速离心，底部沉淀用无菌人间充质干细胞培养基稀释后，进而使用qEV凝胶过滤层析柱过滤，制备出10-100nm的脐带间充质干细胞外泌体，将滤液在离心力为2000g下离心5分钟，沉淀为外泌体，按质量分数比，量取20-45份外泌体以及55-80份蒸馏水，常温下搅拌均匀。
甘草外泌体的制备：
将甘草洗净切碎榨汁，用200-300目的筛子过滤，弃去甘草残渣，采用差速离心法对滤液进行处理，首先在离心力为2000-8000g下，离心20-40分钟，弃去沉淀，再将上清液在离心力为5000-10000g下，离心40-130分钟，弃去沉淀，再将上清液在离心力为10000-200000g下离心30-200分钟，用PBS重悬沉淀，通过密度梯度离心纯化，密度梯度溶液采用蔗糖溶液，在离心力为100000-250000g下离心，时间为120-360分钟，将外泌体吸出，在离心力为50000-300000离心力下离心，时间为30-120分钟，得到外泌体沉淀，按质量分数比，量取30-50份甘草外泌体以及50-70份蒸馏水，常温下搅拌均匀。
外泌体美容液的制备：
按照体积份数比，包括6-16份脐带间充质干细胞外泌体，8-13份甘草外泌体，6-9份香菇多糖，1-2.5份生育酚，1-3份透明质酸钠，56.5-78份无菌培养基，常温下搅拌均匀；按质量分数比，所述脐带间充质干细胞外泌体包括20-45份外泌体以及55-80份蒸馏水，按质量分数比，所述甘草外泌体包括30-50份甘草外泌体以及50-70份蒸馏水。
具体实施方式
以下以具体实施例来说明本发明的技术方案，但本发明的保护范围不限于此：
实施例1
本发明提供了一种外泌体美容液，按体积份数比，包括6份脐带间充质干细胞外泌体，8份甘草外泌体，10份香菇多糖，1份生育酚，1份透明质酸钠，74份无菌培养基。其中间充质干细胞外泌体按质量分数比，包含20份外泌体以及80份蒸馏水，甘草外泌体按质量分数比，包含30份外泌体以及70份蒸馏水。
实施例2
本发明提供了一种外泌体美容液，按体积份数比，包括16份脐带间充质干细胞外泌体，13份甘草外泌体，16份香菇多糖，4.5份生育酚，5份透明质酸钠，45.5份无菌培养基。其中脐带间充质干细胞外泌体按质量分数比，包含45份外泌体以及55份蒸馏水，甘草外泌体按质量分数比，包含50份外泌体以及50份蒸馏水。
实施例3
本发明提供了一种外泌体美容液，按体积份数比，包括13份脐带间充质干细胞外泌体，11份甘草外泌体，14份香菇多糖，2.5份生育酚，3.5份透明质酸钠，56份无菌培养基，常温下混合均匀。其中脐带间充质干细胞外泌体按质量分数比，包含35份外泌体以及65份蒸馏水，甘草外泌体按质量分数比，包含41份外泌体以及59份蒸馏水。
过敏性实验：
首先在100只裸鼠背部涂抹实施例1-3例配比生成的外泌体美容液，检测人员观察裸鼠背部无有红肿、瘙痒、长痘等不良反应；进而选取60名20-50周岁的成年志愿者分为3组进行试验，分别使用上述3组实施例的产品进行过敏性实验，检测人员在测试人员的颈脖上涂抹，经检测人员检测，所有测试人员的颈脖上都未出现红肿、瘙痒、起痘等不良反应。因此该美容液不会对人体产生不良反应。
疗效测试：
选取25-35岁、36-45岁、46-55岁年龄段的女性各60人，每个年龄段的女性继续分成3组，每组20人，分别使用实施例1-3的产品，每隔7天用微针导入一次，每次导入5ml，持续使用4次，测试者对皱纹改善度、提亮肤色程度、毛孔改善度三项进行评分，修复皮肤度为三项的平均分。每一项总分10分，0-3分为无效果，3.1-6分为效果一般，6.1-8分为效果显著，8.1-10分效果非常显著，检测结果如下：
对上述结果进行分析，该外泌体美容液对于改善皱纹、提亮肤色、改善毛孔都有显著的效果。随着年龄的增加，使用美容液的效果越明显，并且实施例3的效果优于实施例1和2，结合表的结果得出实施例3为最佳实施例。
上述实施例为本发明优选的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明所做的改变均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (6)
1.一种外泌体美容液，其特征在于，按照体积份数比，包括6-16份脐带间充质干细胞外泌体，8-13份甘草外泌体，6-9份香菇多糖，1-2.5份生育酚，1-3份透明质酸钠，56.5-78份无菌培养基，常温下搅拌均匀，所述脐带间充质干细胞按照正常培养方法培养得到，脐带间充质干细胞外 泌体通过超高速离心得到，甘草外泌体通过差速离心等得到；按质量分数比，所述脐带间充质干细胞外泌体包括20-45份外泌体以及55-80份蒸馏水，按质量分数比，所述甘草外泌体包括30-50份甘草外泌体以及50-70份蒸馏水。
2.如权利要求1所述一种外泌体美容液，其特征在于按照体积份数比，包括9-14份脐带间充质干细胞外泌体，10-12份甘草外泌体，7-11份香菇多糖，2-3份生育酚，2-4份透明质酸钠，56-70份无菌培养基，所述脐带间充质干细胞按照正常培养方法培养得到，甘草外泌体通过差速离心等技术得到；按质量分数比，所述脐带间充质干细胞外泌体包括25-40份外泌体以及60-75份蒸馏水，按质量分数比，所述甘草外泌体包括35-45份甘草外泌体以及55-65份蒸馏水。
3.如权利要求2所述的一种外泌体美容液，其特征在于，所述脐带间充质干细胞的培养时间为10-13天。
4.如权利要求3所述一种外泌体美容液，其特征在于，所述无菌培养基为无菌人间充质干细胞培养基。
5.如权利要求4所述一种外泌体美容液，其特征在于，该外泌体美容液主要用于提高面部代谢能力、改善面部细纹、提亮肤色。
6.一种外泌体美容液的制备方法，其特征在于，包括以下步骤：
脐带间充质干细胞外泌体的制备：
将脐带从脐带装载瓶中取出，用生理盐水洗涤，并剪成1.5-2厘米的段状，继续用生理盐水洗涤2-3次，取出华氏胶，洗涤后剪碎呈米粒大小，平铺于培养皿，倒置15-30分钟，加入人间充质培养基和添加剂开始培养，3天后换液，之后每隔2-3天换液一次，至细胞达到80-85%融合结束原代培养，用0.25%胰酶消化，镜下观察细胞呈圆形，加入同体积的胎牛血清终止消化，用PBS洗涤后加入人间充质干细胞培养基重悬，按1:2传代，分瓶培养，取第2-3代细胞生存状态比较好的细胞，用无血清培养基培养48h，收集培养上清液，除去细胞碎片，采用100-280分钟离心力为200000g的超速离心，底部沉淀用无菌人间充质干细胞培养基稀释后，进而使用qEV凝胶过滤层析柱过滤，制备出10-100nm的脐带间充质干细胞外泌体，将滤液在离心力为2000g下离心5分钟，沉淀为外泌体，按质量分数比，量取20-45份外泌体以及55-80份蒸馏水，常温下搅拌均匀；
甘草外泌体的制备：
将甘草洗净切碎榨汁，用200-300目的筛子过滤，弃去甘草残渣，采用差速离心法对滤液进行处理，首先在离心力为2000-8000g下，离心20-40分钟，弃去沉淀，再将上清液在离心力为5000-10000g下，离心40-130分钟，弃去沉淀，再将上清液在离心力为10000-200000g下离心30-200分钟，用PBS重悬沉淀，通过密度梯度离心纯化，密度梯度溶液采用蔗糖溶液，在离心力为100000-250000g下离心，时间为120-360分钟，将外泌体吸出，在离心力为50000-300000离心力下离心，时间为30-120分钟，得到外泌体沉淀，按质量分数比，量取30-50份甘草外泌体以及50-70份蒸馏水，常温下搅拌均匀；
外泌体美容液的制备：
按照体积份数比，包括6-16份脐带间充质干细胞外泌体，8-13份甘草外泌体，6-9份香菇多糖，1-2.5份生育酚，1-3份透明质酸钠，56.5-78份无菌培养基，常温下搅拌均匀；所述脐带间充质干细胞外泌体，按质量分数比，包含20-45份外泌体以及55-80份蒸馏水，所述甘草外泌体，按质量分数比，包含30-50份甘草外泌体以及50-70份蒸馏水。
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