CN114010523B - 皮肤抗衰剂及其应用 - Google Patents
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Abstract
本发明提供了一种皮肤抗衰剂及其应用,该皮肤抗衰剂包括siR‑17‑3p agomir,siR‑17‑3p agomir包括如下核苷酸序列:TGGCAGTGTCTTAGCTGGTTGT。本发明与现有的技术相比,能在细胞代谢的层次上对细胞的衰老进行调节,对真皮层中的成纤维细胞进行调控,提高皮肤的抗衰老能力。
Description
技术领域
本发明创造属于化妆品及医学美容技术领域,尤其是涉及皮肤抗衰剂及其应用。
背景技术
目前针对皮肤修复的大部分产品,只能维持皮肤的状况,而不能改善皮肤状况。皮肤的衰老是内源性生理因素和外源性环境因素共同作用的结果。衰老不仅会影响正常皮肤组织结构和生理功能,还会直接影响皮肤的外观。细胞衰老受到多种内外因素的影响,根据引起细胞衰老原因的不同,可将细胞衰老的机制分为复制型衰老和过早型衰老两种。复制型衰老主要依赖于p53-p21信号通路。细胞受到DNA损伤后会磷酸化激活p53,活化的P53可以上调细胞周期蛋白依赖性激酶(CDK)抑制因子p21,后者可以抑制CDK2/cyclinE以及CDK4/cyclinD等细胞周期调节因子的活性,从而使网膜母细胞瘤蛋白pRb转化为非活性形式的低磷酸化或去磷酸化状态,失活的pRb与促进细胞周期的核转录因子E2F结合,使得E2F不能激活细胞周期必需的基因表达,进而使细胞停滞在G0/G1期,无法启动染色体的复制完成增值,从而引发细胞衰老。当细胞发生衰老的机制后,细胞不会立即进行凋亡。衰老的细胞会在组织中存留很长的一段时间。随着衰老细胞在组织中的积累,皮肤就会出现皱纹、色斑等衰老现象。
近年来具备抗衰老的功能制剂一直受到相关学科领域,特别是医疗保健、美容化妆品科学等领域的高度重视和普遍关注。目前市场上已经研制出种类繁多、剂型多样、琳琅满目的皮肤美容抗衰老化妆品。但是目前市场上存在抗衰老的化妆品,都是直接补充胶原蛋白为主要功能。然而,胶原蛋白分子量大,很难穿透皮肤角质层进入真皮组织中。通过往真皮组织中注射胶原蛋白,虽可以使胶原蛋白进入真皮组织中,但依旧没有改变合成胶原蛋白的成纤维细胞的衰老与凋亡,没有从根本上来改善皮肤的衰老与美白。
发明内容
有鉴于此,本发明创造旨在克服现有技术中的缺陷,提出一种皮肤抗衰剂。本发明与现有的技术相比,能在细胞代谢的层次上对细胞的衰老进行调节,从根本上对真皮层中的成纤维细胞进行调控,提高皮肤的抗衰老能力。
为达到上述目的,本发明创造的技术方案是这样实现的:
皮肤抗衰剂,包括siR-17-3p agomir,所述siR-17-3p agomir包括如下核苷酸序列:TGGCAGTGTCTTAGCTGGTTGT(如SEQ ID NO:1所示)。
作为本发明的一种优选技术方案,所述皮肤抗衰剂还包括去乙酰化酶SIRT1和白黎芦醇。
作为本发明的一种优选技术方案,所述皮肤抗衰剂通过调控真皮组织中的成纤维细胞的抗衰老能力来调节皮肤的衰老。
作为本发明的一种优选技术方案,所述皮肤抗衰剂中去乙酰化酶SIRT1、白黎芦醇、siR-17-3p agomir的质量比为(0.1-1):(1-10):(0.1-1)。
作为本发明的一种优选技术方案,所述皮肤抗衰剂还包括美白组分,所述美白组分包括siR-34a-5p agomir,所述siR-34a-5p agomir包括如下核苷酸序列:ACTGCAGTGAAGGCACTTGTAG(如SEQ ID NO:2所示)。
作为本发明的一种优选技术方案,所述美白组分还包括FOXO4-DRI和烟酰胺。
作为本发明的一种优选技术方案,所述皮肤美白通过促进真皮层中衰老的成纤维细胞的凋亡能力,减少真皮层中衰老的成纤维细胞的数量来调节,达到皮肤美白效果。
作为本发明的一种优选技术方案,所述美白组分中FOXO4-DRI、烟酰胺、siR-34a-5p agomir的质量比为(0.1-1):(1-10):(0.1-1)。
本发明还提出一种含有上述皮肤抗衰剂的化妆品或护肤品。
作为本发明的一种优选技术方案,所述化妆品或护肤品还包括保湿剂、抗氧化剂、抗敏剂、润肤剂、防腐剂、螯合剂、抗炎剂、着色剂、增稠剂、乳化剂、中和剂、稳定剂、芳香剂、溶剂中一种或多种。
作为本发明的一种优选技术方案,所述化妆品或护肤品为液体状、乳液状、膏霜状或固体状。
本发明提供的皮肤抗衰剂中,去乙酰化酶SIRT1和白黎芦醇的作用是促进真皮层中成纤维细胞的分裂能力,延迟细胞的分裂周期。siR-17-3p agomir的作用抑制负性调节作用的CKI重要家族成员p21的表达,干扰细胞周期进程,削弱DNA损伤所诱导的细胞周期阻滞,使细胞摆脱生长抑制信号,从G1期阻滞中被释放出来,通过细胞周期限制点,进入信号非依赖状态,从而继续细胞周期进程完成细胞分裂。皮肤抗衰剂中的去乙酰化酶SIRT1、白黎芦醇、siR-17-3p agomi r三者共同使用,增加了细胞的分裂能力,减少因分裂阻滞而衰老的细胞,延迟细胞的分裂周期,从而对皮肤具有抗衰老能力。
皮肤抗衰剂中美白组分能在细胞代谢的层次上对细胞的凋亡进行调节,从根本上对真皮层中的成纤维细胞进行调控,提高皮肤的美白效果。本发明提供的组合物中,FOXO4-DRI和烟酰胺的作用是促进真皮层中衰老的成纤维细胞的凋亡能力,减少真皮层中衰老的成纤维细胞数量。组合物中的siR-34a-5p agonmir的作用是负性抑制SIR T1、E2F等基因的表达从而促进细胞凋亡;p53上调升高miR-34a表达水平,其靶基因SIRT1受siR-34a-5p抑制后,对p53的去乙酰化作用被削弱,从而进一步促进p53及其下游细胞周期调控、凋亡等信号过程。siR-34a-5p通过下调Bcl-2和MYC基因,以及上调P53基因发挥对细胞凋亡的作用。组合物中的FOXO4-DRI、烟酰胺、siR-34a-5p agonmir。三者共同使用,更加增加细胞的凋亡能力,减少真皮成中衰老的成纤维细胞数量,达到皮肤色斑的减少,从而对皮肤具有美白能力。
相对于现有技术,本发明创造具有以下优势:
在本发明的皮肤抗衰剂中,通过去乙酰化酶SIRT1、白黎芦醇、siR-17-3p agonmir三者共同作用,来促进真皮层中成纤维细胞的分裂能力,减少因分裂阻滞而衰老的细胞,延迟成纤维细胞的分裂周期,从而提高皮肤的抗衰老能力。在美白组分中,通过FOXO4-DRI、烟酰胺、siR-34a-5p agonmir三者共同作用,来促进真皮层中成纤维细胞的凋亡能力,减少真皮成中衰老的成纤维细胞数量,达到皮肤色斑的减少,从而对皮肤具有美白能力。
附图说明
图1为CCK-8细胞活性检测标准曲线。
具体实施方式
除有定义外,以下实施例中所用的技术术语具有与本发明创造所属领域技术人员普遍理解的相同含义。以下实施例中所用的实验试剂,如无特殊说明,均为常规生化试剂;所述实验方法,如无特殊说明,均为常规方法。
下面结合实施例来详细说明本发明创造。
一、实验材料
在NCBI数据库和miRBase数据库中查找到siR-17-3p和siR-34a-5p的序列,合成siR-17-3p agomir和siR-34a-5p agomir;其中siR-17-3p agomir的核苷酸序列为TGGCAGTGTCTTAGCTGGTTGT,siR-34a-5p agomir的核苷酸序列为ACTGCAGTGAAGGCACTTGTAG。
去乙酰化酶/SIRT1购自赫澎(上海)生物科技有限公司;
FOXO4-DRI购自无锡海伦生物科技有限公司;
白藜芦醇购自西安博林生物技术有限公司;
烟酰胺购自江西伯业医药科技有限公司。
二、实验方法
1、皮肤抗衰老实验
1.1、细胞培养
取人源的成纤维细胞进行培养,吸弃瓶中旧培养基,像培养瓶中加入适量PBS液覆盖培养瓶细胞生长面,洗涤细胞,弃PBS液。加入0.25%胰蛋白酶消化,常温下消化一段时间,在显微镜下观察,当大部分细胞周边皱缩并变圆时,立即加入适量的DMEM培养基终止消化。吸管吹打瓶壁3遍,所得细胞悬浮液1:3传代培养,24h观察并换液,根据细胞生长情况及时传代。
1.2、SIRT1、白藜芦醇以及siR-17-3p agomir对成纤维细胞增殖的影响
将同一株且同状态培养下的细胞分别接种到13个培养瓶中,分别编号1、2、3、4、5、6、7、8、9、10、11、12、13。以1-12号培养瓶为实验组,按照下表添加不同质量浓度的SIRT1、白藜芦醇和siR-17-3p agomir,以13号培养瓶为空白对照,13号培养瓶中不加任何的SIRT1、白藜芦醇和siR-17-3p agomir,继续培养。
表1 1-13号培养瓶中浓度配比
1.3、CCK-8法检测细胞的增殖情况
将1-13号培养瓶内的细胞用胰酶消化后,以1.5×103个/孔的量接种细胞到96孔板,每孔2×103个/孔,每个实验均设3个复孔,37℃、5%CO2细胞培养箱中培养。向每孔加入100uL的培养基和10uL的CCK-8溶液。将96孔板在培养箱内温育,用酶标仪测定在450nm下测定吸光度,连续计数5天。将每次实验均重复3次,对数据用软件进行统计学分析。CCK-8细胞活性检测标准曲线见图1。
1.4实验结果
由表2的数据可知:SIRT1、白藜芦醇、siR-17-3p agonmir对成纤维细胞的抗衰老具有促进能力,细胞抗衰老率可达43.55%。
具体实验结果见下表:
表2抗衰老实验数据
2、皮肤美白实验
2.1、细胞培养
取人源的成纤维细胞进行培养,吸弃瓶中旧培养基,向培养瓶中加入适量PBS液覆盖培养瓶细胞生长面,洗涤细胞,弃PBS液。加入0.25%胰蛋白酶消化,常温下消化一段时间,在显微镜下观察,当大部分细胞周边皱缩并变圆时,立即加入适量的DMEM培养基终止消化。吸管吹打瓶壁3遍,所得细胞悬浮液1:3传代培养,24h观察并换液,根据细胞生长情况及时传代。
2.2、FOXO4-DRI和烟酰胺以及siR-34a-5p agomir对成纤维细胞的影响
将同一株且经过过氧化氢处理后的衰老细胞分别接种到4个培养瓶中,分别编号1-13。以1-12号培养瓶为实验组,按照下表添加不同质量浓度的FOXO4-DRI、烟酰胺以及siR-34a-5p agomir,以13号培养瓶为空白对照,13号培养瓶中不加任何的FOXO4-DRI、烟酰胺以及siR-34a-5p agomir,继续培养。
表3 1-13号培养瓶中浓度配比
2.3、CCK-8法检测细胞的增殖情况
将1-13号培养瓶内的细胞用胰酶消化后,以1.5×103个/孔的量接种细胞到96孔板,每孔2×103个/孔,每个实验均设3个复孔,37℃、5%CO2细胞培养箱中培养。向每孔加入100uL的培养基和10uL的CCK-8溶液。将96孔板在培养箱内温育,用酶标仪测定在450nm下测定吸光度,连续计数5天。将每次实验均重复3次,对数据用软件进行统计学分析。CCK-8细胞活性检测标准曲线见图1。
2.4实验结果
由表4的数据可知:FOXO4-DRI、烟酰胺、siR-34a agomir对成纤维细胞的凋亡具有促进能力,细胞凋亡率可达26.92%。
具体实验结果见下表:
表4促凋亡实验数据
以上所述仅为本发明创造的较佳实施例而已,并不用以限制本发明创造,凡在本发明创造的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明创造的保护范围之内。
序列表
<110> 天津穗纳生物医药科技有限公司
<120> 皮肤抗衰剂及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 1
tggcagtgtc ttagctggtt gt 22
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence
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actgcagtga aggcacttgt ag 22
Claims (7)
1.皮肤抗衰剂,其特征在于:包括siR-17-3p agomir,所述siR-17-3p agomir为如下核苷酸序列:TGGCAGTGTCTTAGCTGGTTGT;所述皮肤抗衰剂还包括去乙酰化酶SIRT1和白黎芦醇;所述皮肤抗衰剂中去乙酰化酶SIRT1、白黎芦醇、siR-17-3p agomir的质量比为(0.1-1):(1-10):(0.1-1)。
2.根据权利要求1所述的皮肤抗衰剂,其特征在于:所述皮肤抗衰剂还包括美白组分,所述美白组分包括siR-34a-5p agomir,所述siR-34a-5p agomir为如下核苷酸序列:ACTGCAGTGAAGGCACTTGTAG。
3.根据权利要求2所述的皮肤抗衰剂,其特征在于:所述美白组分还包括FOXO4-DRI和烟酰胺。
4.根据权利要求3所述的皮肤抗衰剂,其特征在于:所述美白组分中FOXO4-DRI、烟酰胺、siR-34a-5p agomir的质量比为(0.1-1):(1-10):(0.1-1)。
5.一种含有权利要求1-4任一所述的皮肤抗衰剂的化妆品或护肤品。
6.根据权利要求5所述的护肤品或者化妆品,其特征在于:还包括保湿剂、抗氧化剂、抗敏剂、润肤剂、防腐剂、螯合剂、抗炎剂、着色剂、增稠剂、乳化剂、中和剂、稳定剂、芳香剂、溶剂中一种或多种。
7.根据权利要求5所述的护肤品或者化妆品,其特征在于:所述化妆品或护肤品为液体状、乳液状、膏霜状或固体状。
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