CN114081861A - 一种干细胞外泌体与复合肽抗衰制剂及其制备方法和应用 - Google Patents
一种干细胞外泌体与复合肽抗衰制剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种干细胞外泌体与复合肽抗衰制剂及其制备方法和应用,该制剂包括以下质量份组分:间充质干细胞外泌体0.001~0.005份、间充质干细胞复合肽溶液2.5~12.5份和生理盐水87.5~97.5份。本发明的干细胞外泌体与复合肽抗衰制剂,可有效保证干细胞外泌体的活性,可有效促进成纤维细胞的增殖和多种对皮肤有益成分的生成,对皮肤老化现象有明显改善的作用。
Description
技术领域
本发明属于生物技术领域,具体涉及一种干细胞外泌体与复合肽抗衰制剂及其制备方法和应用。
背景技术
间充质干细胞广泛存在与各类间充质组织,具有自我更新并分化成各种细胞谱系的能力。因此,近年来,间充质干细胞逐渐成为皮肤抗衰领域的研究热点,研究表明其对真皮层多种细胞的增殖有着明显的促进作用。
外泌体是由细胞分泌的一类具有脂质双分子层的球状细胞外囊泡,以往认为它是处理细胞代谢产物的工具。但研究表明,外泌体所携带的蛋白质、核酸、微小RNA等可以在细胞间传递生物学信息,对其他细胞产生调控作用。目前,对外泌体的认知主要是干细胞来源的外泌体对心血管、肝脏以及神经等多器官、系统的再生修复,这显示了干细胞来源外泌体在抗衰老方面的潜力。同时,又有研究表明,间充质干细胞的外泌体同样可以调节和促进真皮层多种细胞的增殖和分泌物质,尤其是促进成纤维细胞的增殖、分泌弹力蛋白和胶原蛋白等,在维持皮肤的弹性和水分中发挥重要作用。相比于间充质干细胞而言,其外泌体作为亚细胞结构,免疫原性低,有更好的耐受性,生物性能也更加稳定。因此,MSC外泌体有望成为MSCs生物学功能的有效替代物,成为皮肤抗衰领域研究的热点。
皮肤组织存在少量的成体干细胞,它具有多向分化的潜能,是皮肤年轻态的储备力量,其衰竭是皮肤衰老的重要原因之一。研究表明,不同组织来源的外泌体都能与成纤维细胞相互作用,促进细胞分泌各种基质蛋白,增强I/II胶原和弹性蛋白的产生,调节细胞的迁移、增殖以及基质层的生成。此外,外泌体还被发现可以通过皮下注射的形式刺激神经酰胺的合成。神经酰胺是增加皮肤角质层水合度,维持皮肤屏障、抗衰老及美白等方面的重要物质,也是如今抗衰老的热门成分。不光如此,还有学者发现,来自人类干细胞的外泌体可以缓解干细胞在培养中的老化,对衰老皮肤成体干细胞有着积极作用。以上结果都表明间充质干细胞外泌体有着良好的预防和治疗皮肤衰老的潜力。
发明内容
针对上述现有技术,本发明提供一种干细胞外泌体与复合肽抗衰制剂及其制备方法和应用。
为了达到上述目的,本发明所采用的技术方案是:提供一种干细胞外泌体与复合肽抗衰制剂,包括以下质量份组分:间充质干细胞外泌体0.001~0.005份、间充质干细胞复合肽溶液2.5~12.5份和生理盐水87.5~97.5份。
进一步,间充质干细胞外泌体由以下步骤制得:
(1)粗提外泌体:将间充质干细胞上清液于2500~3500rpm离心15~25min,收集上清液过0.22μm无菌过膜,将滤液与8w/v%PEG 6000溶液按体积比1~2:1混匀,,其后于4℃、3500~4500rpm离心25~35min,弃上清,再次离心至管壁上的液体离下,其后弃二分之一上清,保留下层液体;
(2)精提外泌体:将下层液体于90000~110000g离心60~80min,去除上清液,沉淀以110000~130000xg超速离心60~80min,弃上清,沉淀用PBS缓冲液重悬后,即得。
进一步,间充质干细胞复合肽溶液由以下步骤制得:
(1)将间充质干细胞上清液用冷冻干燥法脱水,其后加入胰蛋白酶和肽酶于50~60℃水解6~7h,得水解液;
(2)将水解液通过10000分子量膜进行超滤,再取透过液进行凝胶柱层析,分段收取多肽、寡肽和氨基酸组成的峰物质,即得。
进一步,步骤(1)中加入胰蛋白酶和肽酶的质量比为1~5:1。
进一步,间充质干细胞上清液由以下步骤制得:
(1)脐带干细胞预处理:洗净消毒后的脐带剪成2-3cm的小段,并去除脐带上脐动脉、脐静脉和羊膜,得华通氏胶;
(2)干细胞分离:将经预处理的华通氏胶剪成1~3mm的碎片,然后用0.25wt%的胰酶消化30~40min,其后过滤得滤液1,过滤后的脐带组织再用0.25wt%的胰酶消化30~40min,其后过滤得滤液2;合并滤液1和滤液2,加入干细胞培养液终止消化,其后离心弃去上清后,接种于MSC完全培养基中,于37℃、5%CO2、饱和湿度下培养至原代细胞密度达到80%~90%时传代,当细胞密度达1~2×106个/ml,加入生理盐水,离心收集上清液,即得。
进一步,步骤(2)中离心为于2000~3000rpm离心5~10min。
进一步,步骤(2)中过滤为过100um筛网。
本发明还提供了上述干细胞外泌体与复合肽抗衰制剂的制备方法,包括以下步骤:将间充质干细胞外泌体、间充质干细胞复合肽溶液和生理盐水混合均匀,即得。
本发明还提供了上述干细胞外泌体与复合肽抗衰制在制作化妆品中应用。
本发明的有益效果是:
间充质干细胞培养上清中富含大量的生长因子,包括成纤维细胞生长因子、血管生长因子、表皮生长因子等,能够激活皮肤细胞再生,促进胶原蛋白生成,修复表皮,使得皮肤恢复健康和弹性。利用干燥冷冻技术将间充质干细胞培养上清中的水分去除,再用复合酶将大分子蛋白质分解为皮肤能吸收的多肽,所得复合肽能够为细胞提供营养,能够改善皮肤细胞代谢,增强胶原蛋白活性,抑制黑色素生成,达到滋养肌肤的效果。
皮肤抗衰最终的目的是促进成纤维细胞的增殖和多种对皮肤有益成分的生成,外泌体从mRNA基因水平翻译为蛋白质,复合肽成分直接促进成纤维细胞的增殖,二者共同作用能够更好地促进皮肤细胞再生,达到抗衰袪皱的作用。因而,将二者按照一定比例配制的皮肤抗衰制剂能有效提升真皮层组织、细胞、皮肤基底层和表皮层的功能,刺激真皮层胶原蛋白和水分的生成,对皮肤老化现象有明显改善的作用。该制剂可根据不同化妆品的需求,与其进行配伍后生产精华液、润肤膏等各种化妆品。
具体实施方式
下面结合实施例对本发明的具体实施方式做详细的说明。
实施例1
一种干细胞外泌体与复合肽抗衰制剂,包括以下质量份组分:间充质干细胞外泌体0.003份、间充质干细胞复合肽溶液8份和生理盐水92份。
其中,间充质干细胞外泌体由以下步骤制得:
(1)粗提外泌体:将间充质干细胞上清液于3000rpm离心20min,收集上清液过0.22μm无菌过膜,将滤液与8w/v%PEG 6000溶液按体积比1.5:1混匀,,于4℃静置过夜,其后于4℃、4000rpm离心30min,弃上清,再次离心至管壁上的液体离下,其后弃二分之一上清,保留下层液体;
(2)精提外泌体:将下层液体于100000g离心70min,去除上清液,沉淀以120000g超速离心70min,弃上清,沉淀用PBS缓冲液重悬后,最后分装保存于-80℃。
其中,间充质干细胞上清液由以下步骤制得:
(1)脐带干细胞预处理:将脐带用生理盐水清洗去除残血,再用75%酒精消毒30s,其后去除脐带两端各2cm,再将脐带剪成2cm的小段,再次用生理盐水清洗残血,其后去除脐带上脐动脉、脐静脉和羊膜,得华通氏胶,然后浸没在生理盐水的中;
(2)干细胞分离:将经预处理的华通氏胶剪成2mm的大小,再加入0.25wt%胰酶(含EDTA)消化30min,其后过100um筛网得滤液1,过滤后的脐带组织再加入0.25wt%胰酶((含EDTA))消化30min,其后过100um筛网得滤液2;合并滤液1和滤液2,加入干细胞培养液终止消化,其后于2500rpm离心8min弃去上清后,接种于MSC完全培养基中,于37℃、5%CO2、饱和湿度下培养至原代细胞密度达到85%时传代,当细胞密度达1.5×106个/ml时,加入生理盐水,于2500rpm离心8min收集上清液,即得。
其中,间充质干细胞复合肽溶液由以下步骤制得:
(1)将间充质干细胞上清液用冷冻干燥法脱水,其后加入质量比为3:1的胰蛋白酶和肽酶于55℃水解6.5h,得水解液;
(2)将水解液通过10000分子量膜进行超滤,再取透过液进行凝胶柱层析,分段收取多肽、寡肽和氨基酸组成的峰物质,即得。
上述干细胞外泌体与复合肽抗衰制剂的制备方法,包括以下步骤:将间充质干细胞外泌体、间充质干细胞复合肽溶液和生理盐水混合均匀,即得。
实施例2
一种干细胞外泌体与复合肽抗衰制剂,包括以下质量份组分:间充质干细胞外泌体0.005份、间充质干细胞复合肽溶液12.5份和生理盐水87.5份。
其中,间充质干细胞外泌体由以下步骤制得:
(1)粗提外泌体:将间充质干细胞上清液于3500rpm离心15min,收集上清液过0.22μm无菌过膜,将滤液与8w/v%PEG 6000溶液按体积比1:1混匀,于4℃静置过夜,其后于4℃、4500rpm离心25min,弃上清,再次离心至管壁上的液体离下,其后弃二分之一上清,保留下层液体;
(2)精提外泌体:将下层液体于110000g离心60min,去除上清液,沉淀以130000xg超速离心60min,弃上清,沉淀用PBS缓冲液重悬后,最后分装保存于-80℃。
其中,间充质干细胞上清液由以下步骤制得:
(1)脐带干细胞预处理:将脐带用生理盐水清洗去除残血,再用75%酒精消毒30s,其后去除脐带两端各1cm,再将脐带剪成2cm的小段,再次用生理盐水清洗残血,其后去除脐带上脐动脉、脐静脉和羊膜,得华通氏胶,然后浸没在生理盐水的中;
(2)干细胞分离:将经预处理的华通氏胶剪成1mm的大小,再加入0.25wt%胰酶(含EDTA)消化40min,其后过100um筛网得滤液1,过滤后的脐带组织再加入0.25wt%胰酶(含EDTA)消化40min,其后过100um筛网得滤液2;合并滤液1和滤液2,加入干细胞培养液终止消化,其后于2000rpm离心10min弃去上清后,接种于MSC完全培养基中,于37℃、5%CO2、饱和湿度下培养至原代细胞密度达到90%时传代,当细胞密度达2×106个/ml时,加入生理盐水,于2000rpm离心10min收集上清液,即得。
其中,间充质干细胞复合肽溶液由以下步骤制得:
(1)将间充质干细胞上清液用冷冻干燥法脱水,其后加入质量比为5:1的胰蛋白酶和肽酶于60℃水解6h,得水解液;
(2)将水解液通过10000分子量膜进行超滤,再取透过液进行凝胶柱层析、分段收取多肽、寡肽和氨基酸组成的峰物质,即得。
上述干细胞外泌体与复合肽抗衰制剂的制备方法,包括以下步骤:将间充质干细胞外泌体、间充质干细胞复合肽溶液和生理盐水混合均匀,即得。
实施例3
一种干细胞外泌体与复合肽抗衰制剂,包括以下质量份组分:间充质干细胞外泌体0.001份、间充质干细胞复合肽溶液2.5份和生理盐水97.5份。
其中,间充质干细胞外泌体由以下步骤制得:
(1)粗提外泌体:将间充质干细胞上清液于2500rpm离心25min,收集上清液过0.22μm无菌过膜,将滤液与8w/v%PEG 6000溶液按体积比2:1混匀,于4℃静置过夜,其后于4℃、3500rpm离心35min,弃上清,再次离心至管壁上的液体离下,其后弃二分之一上清,保留下层液体;
(2)精提外泌体:将下层液体于90000g离心80min,去除上清液,沉淀以110000xg超速离心80min,弃上清,沉淀用PBS缓冲液重悬后,最后分装保存于-80℃。
其中,间充质干细胞上清液由以下步骤制得:
(1)脐带干细胞预处理:将脐带用生理盐水清洗去除残血,再用75%酒精消毒30s,其后去除脐带两端各1cm,再将脐带剪成3cm的小段,再次用生理盐水清洗残血,其后去除脐带上脐动脉、脐静脉和羊膜,得华通氏胶,然后浸没在生理盐水的中;
(2)干细胞分离:将经预处理的华通氏胶剪成3mm的大小,再加入0.25wt%胰酶(含EDTA)消化35min,其后过100um筛网得滤液1,过滤后的脐带组织再加入0.25wt%胰酶(含EDTA)消化35min,其后过100um筛网得滤液2;合并滤液1和滤液2,加入干细胞培养液终止消化,其后于3000rpm离心5min弃去上清后,接种于MSC完全培养基中,于37℃、5%CO2、饱和湿度下培养至原代细胞密度达到80%时传代,当细胞密度达1×106个/ml时,加入生理盐水,于3000rpm离心5min收集上清液,即得。
其中,间充质干细胞复合肽溶液由以下步骤制得:
(1)将间充质干细胞上清液用冷冻干燥法脱水,其后加入质量比为1:1的胰蛋白酶和肽酶于50℃水解7h,得水解液;
(2)将水解液通过10000分子量膜进行超滤,再取透过液进行凝胶柱层析、分段收取多肽、寡肽和氨基酸组成的峰物质,即得。
上述干细胞外泌体与复合肽抗衰制剂的制备方法,包括以下步骤:将间充质干细胞外泌体、间充质干细胞复合肽溶液和生理盐水混合均匀,即得。
对比例1
将实施例1中的间充质干细胞外泌体去掉,其余与实施例1相同。
对比例2
将实施例1中的间充质干细胞复合肽溶液去掉,其余与实施例1相同。
对比例3
将实施例1中的间充质干细胞复合肽溶液改为胶原蛋白肽溶液,其余与实施例1相同。
下面测试实施例与对比例中干细胞外泌体与复合肽抗衰制剂的抗衰作用:
试验方法:筛选250例皮肤出现明显皱纹的女性受试志愿者,年龄30~50岁,平均年龄40岁,随机平均分为5组,分别为对照组、实施例1、2、3组、对比例1组,早、晚洁面后取适量的精华液涂抹在面部,在试验期间避免日晒,或使用SPF≥30的防晒产品;在试验期间不使用其他抗衰产品。其中,对照组使用市场上在售的抗衰产品,实施例1、2、3组和对比例1组分别使用本发明实施例1、实施例2和实施例3、对比例1的抗衰产品。试验时间为90天,记录试验期间发生的不良反应,并对受试者进行效果评价。
皮肤皱纹变化情况评估:通过观察受试者的脸部皱纹变化情况来评价。
评判标准:
优:细纹完全消失,深度皱纹明显变浅;
良:细纹数量减少,深度皱纹有所减轻;
中:细纹有所减轻,深度皱纹稍有减轻。
差:细纹数量不变,深度皱纹无好转。
使用感评估:主要评价使用产品后,受试者是否出现过敏、干燥、油腻等不适反应,并在第90天对受试者采用无创性仪器检测受试脸部的含水量、油脂含量,进行统计分析。
试验结果:如表1、表2、表3所示。
表1皮肤皱纹变化情况
优(例) | 良(例) | 中(例) | 差(例) | |
对照组 | 22 | 13 | 10 | 5 |
实施例1组 | 31 | 17 | 2 | 0 |
实施例2组 | 33 | 14 | 3 | 0 |
实施例3组 | 29 | 20 | 1 | 0 |
对比例1组 | 21 | 17 | 8 | 4 |
对比例2组 | 18 | 15 | 10 | 7 |
对比例3组 | 20 | 16 | 8 | 6 |
表2:使用感评估
表3:角质层含水量和油脂含量测量结果
虽然结合实施例对本发明的具体实施方式进行了详细地描述,但不应理解为对本专利的保护范围的限定。在权利要求书所描述的范围内,本领域技术人员不经创造性劳动即可作出的各种修改和变形仍属本专利的保护范围。
Claims (9)
1.一种干细胞外泌体与复合肽抗衰制剂,其特征在于,包括以下质量份组分:间充质干细胞外泌体0.001~0.005份、间充质干细胞复合肽溶液2.5~12.5份和生理盐水87.5~97.5份。
2.根据权利要求1所述的干细胞外泌体与复合肽抗衰制剂,其特征在于,所述间充质干细胞外泌体由以下步骤制得:
(1)粗提外泌体:将间充质干细胞上清液于2500~3500rpm离心15~25min,收集上清液过0.22μm无菌过膜,将滤液与8w/v%PEG 6000溶液按体积比1~2:1混匀,于4℃静置过夜,其后于4℃、3500~4500rpm离心25~35min,弃上清,再次离心至管壁上的液体离下,其后弃二分之一上清,保留下层液体;
(2)精提外泌体:将下层液体于90000~110000g离心60~80min,去除上清液,沉淀以110000~130000xg超速离心60~80min,弃上清,沉淀用PBS缓冲液重悬后,即得。
3.根据权利要求1所述的干细胞外泌体与复合肽抗衰制剂,其特征在于,所述间充质干细胞复合肽溶液由以下步骤制得:
(1)将间充质干细胞上清液用冷冻干燥法脱水,其后加入胰蛋白酶和肽酶于50~60℃水解6~7h,得水解液;
(2)将水解液通过10000分子量膜进行超滤,再取透过液进行凝胶柱层析,分段收取多肽、寡肽和氨基酸组成的峰物质,即得。
4.根据权利要求3所述的干细胞外泌体与复合肽抗衰制剂,其特征在于:步骤(1)中加入胰蛋白酶和肽酶的质量比为1~5:1。
5.根据权利要求2或3所述的干细胞外泌体与复合肽抗衰制剂,其特征在于,所述间充质干细胞上清液由以下步骤制得:
(1)脐带干细胞预处理:将洗净消毒后的脐带剪成2-3cm的小段,并去除脐带上脐动脉、脐静脉和羊膜,得华通氏胶;
(2)干细胞分离:将经预处理的华通氏胶剪成1~3mm的碎片,然后用0.25wt%的胰酶消化30~40min,其后过滤得滤液1,过滤后的脐带组织再用0.25wt%的胰酶消化30~40min,其后过滤得滤液2;合并滤液1和滤液2,加入干细胞培养液终止消化,其后离心弃去上清后,接种于MSC完全培养基中,于37℃、5%CO2、饱和湿度下培养至原代细胞密度达到80%~90%时传代,当细胞密度达1~2×106个/ml时,加入生理盐水,离心收集上清液,即得。
6.根据权利要求5所述的干细胞外泌体与复合肽抗衰制剂,其特征在于:步骤(2)中离心为于2000~3000rpm离心5~10min。
7.根据权利要求5所述的干细胞外泌体与复合肽抗衰制剂,其特征在于:步骤(2)中过滤为过80目筛网。
8.根据权利要求1~7所述干细胞外泌体与复合肽抗衰制剂的制备方法,其特征在于,包括以下步骤:将间充质干细胞外泌体、间充质干细胞复合肽溶液和生理盐水混合均匀,即得。
9.根据权利要求1所述的干细胞外泌体与复合肽抗衰制剂在制作化妆品中应用。
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