CN114159374A - 用于皮肤抗衰老间充质干细胞外泌体组合物的制备方法及产品和应用 - Google Patents
用于皮肤抗衰老间充质干细胞外泌体组合物的制备方法及产品和应用 Download PDFInfo
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Abstract
本发明公开了一种用于皮肤抗衰老的脐带来源的间充质干细胞外泌体组合物的制备方法及其产品和应用,脐带来源的间充质干细胞的培养:正常进行细胞培养、消化、传代;脐带来源的间充质干细胞外泌体的获取:收集脐带来源的间充质干细胞培养液上清,进行离心和超滤浓缩,得干细胞外泌体;抗氧化剂的混合:脐带来源的间充质干细胞外泌体与抗氧化剂物理混合。本发明所述脐带来源的间充质干细胞外泌体组合物中含有脐带来源的间充质干细胞外泌体和抗氧化剂,具有降低细胞内活性氧含量,促进细胞增殖及抗细胞衰老的作用,可在抗衰老方向的化妆品中应用。
Description
技术领域
本发明涉及一种脐带来源的间充质干细胞外泌体及其用途,具体涉及一种用于皮肤抗衰老的脐带来源的间充质干细胞干细胞外泌体组合物的制备方法及其产品和应用。
背景技术
外泌体(Exosome)是由细胞内囊泡结构释放到细胞外的一种膜性囊泡,直径约40—160nm(平均直径为100nm)。由于质膜和多囊泡体之间融合的结果,外泌体样囊泡通过胞吐作用被分泌到胞外空间。外泌体样囊泡被脂质双层分为内部和外部,可以包含细胞的许多成分,包括DNA、RNA、脂质、代谢物、细胞质和细胞表面蛋白。外泌体的主要功能是进行细胞间信息交流,一般是通过传递膜表面或腔内的生物分子来进行细胞间信号传递,从而调节细胞的正常生理功能。外泌体的组成成分均来源于细胞,避免了人工合成材料的使用带来的细胞毒性、生物相容性等问题。由于外泌体继承了细胞的磷脂、表面蛋白等物质,具有丰富的生理功能,可以提高有效成分如抗氧化剂的传递效率。
皮肤衰老是人体衰老固有的生理机制和外界风吹日晒、环境污染等损害共同作用的结果。皮肤衰老通常体现在皮肤弹性降低和皮肤皱纹的产生。据报道,胶原蛋白和弹性蛋白的减少将导致皮肤弹性降低和皮肤皱纹产生,胶原蛋白和弹性蛋白受胶原蛋白酶和弹性蛋白酶调控,胶原蛋白酶和弹性蛋白酶能够引起胶原蛋白和弹性蛋白降解,从而导致胶原蛋白和弹性蛋白减少,导致皱纹产生,皮肤弹性减低。
发明内容
本发明目的在于提供一种用于皮肤抗衰老的脐带来源的间充质干细胞外泌体组合物的制备方法。
本发明的再一目的在于:提供一种上述方法制备的用于皮肤抗衰老的脐带来源的间充质干细胞外泌体组合物产品。
本发明的又一目的在于:提供一种上述产品的应用。
本发明目的通过以下方案实现:一种用于皮肤抗衰老的脐带来源的间充质干细胞外泌体组合物的制备方法,包括脐带来源的间充质干细胞的培养、外泌体提取、与抗氧化剂混合,包括以下步骤:
(1)脐带来源的间充质干细胞的培养:进行细胞培养、消化、传代;
(2)脐带来源的间充质干细胞外泌体的获取:收集脐带来源的间充质干细胞培养液上清,进行离心和超滤浓缩,得干细胞外泌体;
(3)抗氧化剂的混合:脐带来源的间充质干细胞外泌体与抗氧化剂按质量比为1:3~5:1物理混合,所述抗氧化剂包括花青素、白藜芦醇、维生素A、维生素C、维生素E、类胡萝卜素成分中的一种或几种。
在上述方案基础上,优选的,收集传代次数为10代以内的细胞。
优选的,选择2~10代细胞是由于低代数的原代细胞具有更好的细胞因子分泌功能。
将代次在2~10代内的脐带来源的间充质干细胞按照1×105个/ml的密度进行正常培养,细胞融合度达到80%以上时,更换新鲜的不含抗生素的无血清培养基,培养3~5d后收集细胞培养上清,即条件培养基。所述条件培养基在4℃,100~300g条件下离心5~10min,弃去沉淀杂质,收集上层液体,用2000g离心20min,收集上层液体,再通过超滤膜进行超滤浓缩,去除小分子,最后将所得液体用0 .22μm无菌过滤器过滤除菌,获得外泌体。
优选的,所述的超滤膜为100000NW CO超滤膜。
培养基不含动物源血清。
本发明中,外泌体溶液和抗氧化剂的。脐带来源的间充质干细胞外泌体和抗氧化剂采用质量比为1:3~5:1的配比能更好地促进抗氧化剂的吸收,抵抗氧化应激衰老的效果更佳。
本发明还提供了一种用于皮肤抗衰老的脐带来源的间充质干细胞外泌体组合物,根据上述任一所述方法制备得到,包含了脐带来源的间充质干细胞外泌体和抗氧化剂。该组合物可促进氧化受损的真皮成纤维细胞恢复增殖,进而促进胶原分泌,淡化皱纹;并且脐带来源的间充质干细胞外泌体还能在一定程度上促进抗氧化剂的吸收,使抗氧化剂在皮肤中的利用率更高,能有效对抗皮肤氧化应激衰老。
本发明也提供了一种脐带来源的间充质干细胞外泌体组合物的应用,用于制备皮肤抗衰老化妆品所述化妆品,如水剂、乳剂、膏霜、凝胶或粉剂等化妆品。
所述脐带来源的间充质干细胞外泌体组合物在化妆品中的含量为1~20%。
使用方法为涂敷。
本发明的目的在于提供所述脐带来源的间充质干细胞外泌体组合物在抗衰老化妆品中的应用,包括除皱,紧致等功效。所述化妆品可以为水剂、乳剂、膏霜、凝胶或粉剂。
所述脐带来源的间充质干细胞外泌体组合物能够阻断氧化应激衰老进程,降低氧化受损的真皮成纤维细胞的胞内活性氧含量,提高细胞增殖能力,从而改善皮肤皱纹,减少衰老相关色素沉积。并且脐带来源的间充质干细胞外泌体还能在一定程度上促进抗氧化剂的吸收,提高抗氧化剂在皮肤中的利用率,从而更好的抵抗氧化应激所致皮肤衰老。
一种用于皮肤抗衰老的脐带来源的间充质干细胞外泌体组合物,本发明所述脐带来源的间充质干细胞外泌体组合物中含有脐带来源的间充质干细胞外泌体和抗氧化剂,具有降低细胞内活性氧含量,促进细胞增殖及抗细胞衰老的作用,可在抗衰老方向的化妆品中应用。
一种用于皮肤抗衰老的脐带来源的间充质干细胞外泌体组合物,可促进氧化受损的真皮成纤维细胞恢复增殖,进而促进胶原分泌,淡化皱纹。并且脐带来源的间充质干细胞外泌体还能在一定程度上促进抗氧化剂的吸收,使抗氧化剂在皮肤中的利用率更高,能有效对抗皮肤氧化应激衰老。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用原料均为市售商品。
为更好的说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。
本发明所述一种用于皮肤抗衰老的脐带来源的间充质干细胞外泌体组合物,制备过程包括干细胞的养、外泌体获取,与抗氧化剂混合。具体步骤如下:
(1)细胞培养
将购自市场的P2脐带来源的间充质干细胞,按照1×105个/ml的接种密度进行正常培养,传代次数控制在P10以内,直至收集到足够的细胞,收集培养液上清,所用培养基不含抗生素和动物源血清;
(2)外泌体获得
将收集获得的培养液上清在4℃,100g条件下离心5min,弃去沉淀杂质,收集上层液体,用2000g离心20min,收集上层液体,再通过100000NWCO超滤膜进行超滤浓缩,去除小分子,最后将所得液体用0 .22μm无菌过滤器过滤除菌,获得外泌体。
(3) 外泌体与抗氧化剂混合
步骤(2)所得外泌体溶液,与花青素、白藜芦醇、维生素C等抗氧化剂物理混合。
干细胞外泌体和抗氧化剂的质量比为1:1,其中花青素、白藜芦醇、维生素C的质量比为4:1:1。
Claims (10)
1.一种用于皮肤抗衰老的脐带来源的间充质干细胞外泌体组合物的制备方法,其特征在于,包括以下步骤:
(1)脐带来源的间充质干细胞的培养:进行细胞培养、消化、传代;
(2)脐带来源的间充质干细胞外泌体的获取:收集脐带来源的间充质干细胞培养液上清,进行离心和超滤浓缩,得干细胞外泌体;
(3)抗氧化剂的混合:脐带来源的间充质干细胞外泌体与抗氧化剂按质量比为1:3~5:1物理混合,所述抗氧化剂包括花青素、白藜芦醇、维生素A、维生素C、维生素E、类胡萝卜素成分中的一种或几种。
2.根据权利要求1所述的用于皮肤抗衰老的脐带来源的间充质干细胞外泌体组合物的制备方法,其特征在于,收集传代次数为10代以内的细胞。
3.根据权利要求1或2所述的用于皮肤抗衰老的脐带来源的间充质干细胞外泌体组合物的制备方法,其特征在于,将代次在2~10代内的脐带来源的间充质干细胞按照1×105个/ml的密度进行正常培养,细胞融合度达到80%以上时,更换新鲜的不含抗生素的无血清培养基,培养3~5d后收集细胞培养上清,即条件培养基;所述条件培养基在4℃,100~300g条件下离心5~10min,弃去沉淀杂质,收集上层液体,用2000g离心20min,收集上层液体,再通过超滤膜进行超滤浓缩,去除小分子,最后将所得液体用0 .22μm无菌过滤器过滤除菌,获得外泌体。
4.根据权利要求3所述的用于皮肤抗衰老的脐带来源的间充质干细胞外泌体组合物的制备方法,其特征在于,培养基不含动物源血清。
5.根据权利要求3所述的用于皮肤抗衰老的脐带来源的间充质干细胞外泌体组合物的制备方法,其特征在于,超滤膜为100000NW CO超滤膜。
6.根据权利要求3或4或5任一项所述的用于皮肤抗衰老的脐带来源的间充质干细胞外泌体组合物的制备方法,其特征在于,
(1)细胞培养:将购自市场的P2脐带来源的间充质干细胞,按照1×105个/ml的接种密度进行正常培养,传代次数控制在P10以内,直至收集到足够的细胞,收集培养液上清,所用培养基不含抗生素和动物源血清;
(2)外泌体获得:将收集获得的培养液上清在4℃,100g条件下离心5min,弃去沉淀杂质,收集上层液体,用2000g离心20min,收集上层液体,再通过100000NWCO超滤膜进行超滤浓缩,去除小分子,最后将所得液体用0 .22μm无菌过滤器过滤除菌,获得外泌体;
(3) 外泌体与抗氧化剂混合:
步骤(2)所得外泌体溶液,与花青素、白藜芦醇、维生素C抗氧化剂按质量比为1:1进行物理混合,所述的抗氧化剂为花青素、白藜芦醇、维生素C的质量比为4:1:1的混合物。
7.一种用于皮肤抗衰老的脐带来源的间充质干细胞外泌体组合物,其特征在于根据权利要求1-6任一所述的方法制备得到,包含了脐带来源的间充质干细胞外泌体和抗氧化剂。
8.一种根据权利要求7所述脐带来源的间充质干细胞外泌体组合物的应用,用于制备包括水剂、乳剂、膏霜、凝胶或粉剂的皮肤抗衰老化妆品。
9.根据权利要求8所述脐带来源的间充质干细胞外泌体组合物的应用,其特征在于,所述脐带来源的间充质干细胞外泌体组合物在化妆品中的含量为1~20%。
10.根据权利要求8或9所述脐带来源的间充质干细胞外泌体组合物的应用,其特征在于,使用方法为涂敷。
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