CN117045577A - 一种皮肤护理液及其制备方法与应用 - Google Patents
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Abstract
本发明公开了一种皮肤护理液及其制备方法与应用。本发明皮肤护理液包括脐带间充质干细胞来源的胞外囊泡浓缩液A、基底液B;胞外囊泡浓缩液A中干细胞囊泡的浓度为1×109~10/mL,胞外囊泡浓缩液A、基底液B的体积比为0.1~50:1,基底液B包括以下按体积份计:1~45份干细胞提取物、1~10份甘油三醇、1~60份人血清白蛋白、1~50份氮酮、0.1~30份维生素E、0.1~60份卵磷脂、0.1~30份胆固醇、0.1~30份玻尿酸、0.1~10份吐温、0.1~10份山茶油。本发明所得皮肤护理液用于制备修复腹部皮肤的妊娠纹的护肤品,该产品也可用于制备皮肤II度烧伤及疤痕修复的药物,效果显著。
Description
技术领域
本发明属于皮肤护理技术领域,尤其涉及一种皮肤护理液及其制备方法与应用。
背景技术
妊娠纹的形成主要受妊娠期荷尔蒙的影响,加之腹部膨隆至使皮下弹力纤维与胶原纤维损伤或断裂,腹部皮肤变薄,出现一些宽窄不同、长短不一的粉红色或紫红色的波浪状花纹。分娩后,这些花纹会逐渐消失,留下白色或银白色的有光泽的瘢痕线纹,即妊娠纹。妊娠纹主要出现在腹壁上,也可能出现在大腿内外侧、臀部、胸部、后腰部及手臂等处。初产妇的妊娠纹最为明显,一旦出现妊娠纹就不易消失,严重影响了妇女产后的体态和身心健康。
妊娠纹初期表现为“红色纹”阶段(淡红或紫红色),该阶段相对容易修复,一般出现在产后6个月以内,部分患者甚至能够自愈;多数患者,其妊娠纹在产后6~12月内,一般情况下会逐渐进展为“暗红色+褐色纹”形态,此时肌肤自愈能力显著减弱;随即会进一步恶化,逐渐变白进入“难治愈型”阶段,10年以上难治愈妊娠纹,均为“银色纹+白凹纹”形态,多数情况会伴随患者终身无法去除,目前市面尚无有效产品或技术加以治疗。
妊娠纹主要因真皮层纤维蛋白断裂及后续色素沉着形成断裂纹,因此促进真皮层细胞的再生是修复之关键。传统化学产品多以润滑、保湿、滋润为主,无细胞修复功能;部分含有修复因子的产品因成分单一、含量低、易失活、分子量大难吸收等缺点难以达到预期功效。
脐带组织内含有丰富的间充质干细胞(MSC),取材方便,免疫原性低,目前已证明MSC可以通过旁分泌机制或以囊泡包裹形式分泌大量细胞因子作用于受损部位,进而促进组织修复,也被用于皮肤损伤的修复及美容护肤。
胞外囊泡是一种由不同细胞在正常或病理状态下分泌的微小囊泡,直径在30~150nm,具有脂质双层膜结构。胞外囊泡主要参与细胞通讯和维持组织稳态等生物学功能,干细胞来源的外囊泡在组织再生方面具有良好的临床应用前景,并且具有良好的生物兼容性,为其在医美领域的应用奠定了重要的生物学理论基础。
有研究表明脐带提取的胞外囊泡通过膜融合或者内吞作用后将生物活性物质转运至靶细胞,介导细胞间通讯并影响靶细胞功能,还具有抑制炎症反应、免疫调节、血管生成、参与组织修复的功能,具有稳定、安全、保存方便等特性。
发明内容
本发明的首要目的在于提供一种皮肤护理液。
本发明的再一目的在于提供上述皮肤护理液的制备方法。
本发明的另一目的在于提供上述皮肤护理液的应用。
本发明是这样实现的,一种皮肤护理液,该皮肤护理液包括脐带间充质干细胞来源的胞外囊泡浓缩液A以及基底液B;所述胞外囊泡浓缩液A中干细胞囊泡的浓度为1×109 ~10/mL,所述胞外囊泡浓缩液A、基底液B的体积比为0.1~50:1;
所述基底液B包括以下按体积份计:
本发明进一步公开了上述皮肤护理液的制备方法,该方法包括以下步骤:
(1)培养脐带间充质干细胞;
(2)从脐带间充质干细胞培养物中提取胞外囊泡浓缩液A,4℃保存;
(3)配制基底液B;
(4)在使用时,将所述胞外囊泡浓缩液A作为皮肤护理液或与基底液B混合搭配为皮肤护理液。
优选地,在步骤(1)中,所述脐带间充质干细胞的培养过程为:
采集足月剖宫产新生儿脐带剪成2~3cm小段,清洗后剥剪至1mm2小块,均匀铺于培养瓶中,置于37℃、5%CO2浓度的培养箱中培养,8~24h内加入15mL完全培养基覆盖组织块,培养至第五天半换液,此后每隔2~3天换液;
细胞融合度达到70~80%时,吸出组织块加到一个新的培养瓶中,补加培养基到15mL,1~2天后观察新的细胞从组织块周围爬出,融合度达到70%~80%时进行传代,记为P1;
持续传代操作,获得二次贴壁细胞的P2代到P5代作为备用的脐带间充质干细胞。
优选地,步骤(2)中的胞外囊泡浓缩液A以及步骤(3)基底液B中的干细胞提取物的制备包括以下步骤:
将生长状态良好的5×106脐带间充质干细胞接种至细胞培养皿中,加入无血清的商品化干细胞培养基,细胞的融合度达到80%~90%时,收集培养上清液1;
将上清液1在4℃、以2000g离心8~10min去除完整细胞和死细胞碎片,获得上清液2;
将上清液2在4℃、以10000g离心10min,用0.22μm滤器过滤,得到滤出液1;
将滤出液1使用100kD超滤柱进行浓缩,得到含干细胞外囊泡浓度为1×109~10/mL的胞外囊泡浓缩液A,同时获得非浓缩状态的滤出液2;
将滤出液2使用10kD超滤柱再次进行浓缩,得到干细胞提取物。
本发明进一步公开了上述皮肤护理液在制备修复妊娠纹的护肤品中的应用。
本发明进一步公开了上述皮肤护理液在制备皮肤II度烧伤及疤痕修复药物中的应用。
本发明克服现有技术的不足,提供一种皮肤护理液及其制备方法与应用。本发明皮肤护理液包括脐带间充质干细胞来源的胞外囊泡浓缩液A、基底液B;其中,胞外囊泡浓缩液A中,包括但不限于干细胞囊泡,囊泡内含活性因子,具有促进真皮层细胞生长、修复真皮层细胞损伤等作用;基底液B的各组分主要是保湿、消炎、滋润、促进有效活性成分穿透表皮到达皮下部位的作用,同时也包含部分干细胞来源的游离活性因子成分,即干细胞提取物,而基底液B的保湿、滋润作用又可以促进干细胞囊泡在皮肤表层及真皮层的均匀分布,有利于皮肤的吸收、利用。
在本发明中,基底液B中各组分的主要具体功能表现为:甘油三醇的作用是:保湿,润滑;氮酮的作用是:保湿滋润、抗氧化、舒缓镇静、促吸收、抗菌消炎;维生素E的作用是:抗氧化、修复再生、抗炎镇静、促吸收、提亮肤色;卵磷脂的作用是:促进成分渗透、保湿、锁水、滋润、修复皮肤屏障;胆固醇的作用是:保湿滋润、柔软光滑肌肤、抗炎、增强皮肤弹性、促进成分渗透和抗氧化;玻尿酸的作用是:保湿补水、增加皮肤弹性、减少细纹和皱纹、改善肤色和肤质以及修复受损皮肤;吐温的作用是:乳化稳定、增稠和调节质地、渗透促进、调节表面张力;山茶油的作用是:保湿滋润、抗氧化、抗炎镇静、修复再生、抗菌;
本发明所得皮肤护理液用于制备修复腹部皮肤的妊娠纹的护肤品,该产品也可用于制备皮肤II度烧伤及疤痕修复的药物。
相比于现有技术的缺点和不足,本发明具有以下有益效果:
(1)本发明通过超滤得到脐带间充质干细胞来源的胞外囊泡浓缩液A,最大限度保留了促进细胞生长的营养成分、RNA(核糖核酸)、酶和活性因子等活性物质,去除活细胞、大分子的蛋白、细胞碎片等难吸收成分;基底液B中含有干细胞来源的非囊泡游离活性因子浓缩组分及各类辅助成分;该胞外囊泡浓缩液A与基底液B进行科学配比使用,具有多重功效,包括消炎、保湿、润滑等,达到愈伤、修复、滋润的多重功效,并能有效促进难治性的妊娠纹修复;
(2)本发明皮肤护理液还能够有效实现II度烧伤创面修复,及创面部位的疤痕组织、皱纹和皮肤问题的修复,效果显著。
附图说明
图1是本发明应用实施例1中皮肤护理液修复妊娠纹的效果对比图;其中,图a是修复前,图b是修复后;
图2是本发明应用实施例2中皮肤护理液治疗II度烧伤患者的效果图;其中,图a是治疗前,图b是治疗1d后,图c是治疗6d后,图d是治疗12d后,图e是修复90d后。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1
(1)采集足月剖宫产新生儿脐带剪成2~3cm小段,清洗后剥剪至1mm2小块,均匀铺于培养瓶中,置于37℃、5%CO2浓度的培养箱中培养,8~24h内加入15mL完全培养基覆盖组织块,培养至第五天半换液,此后每隔2~3天换液;
(2)细胞融合度达到70~80%时,吸出组织块加到一个新的培养瓶中,补加培养基到15mL,1~2天后观察新的细胞从组织块周围爬出,融合度达到70%~80%时进行传代,记为P1;
(3)持续传代操作,获得二次贴壁细胞的P2代到P5代作为备用的脐带间充质干细胞。
实施例2
(1)将生长状态良好的5×106脐带间充质干细胞接种至细胞培养皿中,加入无血清的商品化干细胞培养基,细胞的融合度达到80%~90%时,收集培养上清液1;
(2)将上清液1在4℃、以2000g离心8~10min去除完整细胞和死细胞碎片,获得上清液2;
(3)将上清液2在4℃、以10000g离心10min,用0.22μm滤器过滤,得到滤出液1;
(4)将滤出液1使用100kD超滤柱进行浓缩,得到含干细胞外囊泡浓度为1×109~10/mL的胞外囊泡浓缩液A,短期-4℃储存,或-80~-20℃长期储存,同时获得非浓缩状态的滤出液2;
(5)将滤出液2使用10kD超滤柱再次进行浓缩,获得二次浓缩液,该二次浓缩液即为干细胞提取物,用于配制基底液B,同时获得二次滤出液3,二次滤出液3为废液,经无害化处理后丢弃。
实施例3
将1体积份甘油三醇、1体积份上述实施例2制备的干细胞提取物、1体积份人血清白蛋白、1体积份氮酮、0.1体积份维生素E、60体积份卵磷脂、30体积份胆固醇、30体积份玻尿酸、10体积份吐温、10体积份山茶油混合,得到基底液B1。
实施例4
将10体积份甘油三醇、45体积份上述实施例2制备的干细胞提取物、60体积份人血清白蛋白、50体积份氮酮、30体积份维生素E、60体积份卵磷脂、30体积份胆固醇、30体积份玻尿酸、0.1体积份吐温、10体积份山茶油混合,得到基底液B2。
实施例5
将5体积份甘油三醇、20体积份上述实施例2制备的干细胞提取物、30体积份人血清白蛋白、25体积份氮酮、15体积份维生素E、30体积份卵磷脂、15体积份胆固醇、15体积份玻尿酸、5体积份吐温、5体积份山茶油混合,得到基底液B3。
应用实施例1
在本应用实施例中,选择十年以上“难治愈妊娠纹”患者为临床研究对象进行修复治疗,具体过程如下:
(1)在需要使用时,将实施例2低温保存的胞外囊泡浓缩液A取出,与实施例3中制备的基底液B按体积比0.1:1混合,得到皮肤护理液;
(2)将2~3mL皮肤护理液挤于手心,轻拍至腹部妊娠纹处,轻轻按摩至皮肤护理液被皮肤完全吸收(或者可配合物理低温导入装置使用,通过低温探头蘸取皮肤护理液,轻轻在腹部按摩至完全吸收),每天一次持续二十天以上。
结果如图2所示,图1a为治疗前该患者腹部难治性妊娠纹状态,图1b为连续使用本发明皮肤护理液20天后,同样部位妊娠纹改善状态;经对比显示,本发明能够有效改善难治性妊娠纹,使患者皮肤基本恢复至正常状态。
应用实施例2
在本应用实施例中,选择II度烧伤患者为临床对象进行修复治疗,该患者的烧伤创面表现为3CM2水泡并不慎破裂(如图2a所示),过程分为治疗阶段、修复阶段,具体如下:
(1)治疗阶段
将实施例2低温保存的胞外囊泡浓缩液A取出,在该患者烧伤后及时涂抹胞外囊泡(200微升),治疗24h后创面结痂(图2b所示),治疗6d后炎症消失(图2c),待治疗12天结痂脱落(图2d所示);
(2)修复阶段
随后开始每日涂抹皮肤护理液(每次需要使用时,将实施例2低温保存的胞外囊泡浓缩液A取出并与实施例4中制备的基底液B2按体积比50:1混合,修复至90d后疤痕完全消失(如图2e所示)。
综合上述治疗效果可见,本发明脐带间充质干细胞来源的胞外囊泡浓缩液A能够有效促进破损性创面愈合;而脐带间充质干细胞来源的胞外囊泡浓缩液A与基底液B的配合,不仅能修复难治性妊娠纹,而且还能够对烧伤后形成的疤痕实现有效修复。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等;以及本发明所述基底液B采用现有一种或多种组分组合,或进一步添加但不限于玫瑰花露、金缕梅、绿茶、洋甘菊、薰衣草提取液等各类调色剂、增味剂、防腐剂、中草药提取物等辅助成分,均应包含在本发明的保护范围之内。
Claims (6)
1.一种皮肤护理液,其特征在于,该皮肤护理液包括脐带间充质干细胞来源的胞外囊泡浓缩液A以及基底液B;所述胞外囊泡浓缩液A中干细胞囊泡的浓度为1×109~10/mL,所述胞外囊泡浓缩液A、基底液B的体积比为0.1~50:1;
所述基底液B包括以下按体积份计:
2.权利要求1所述的皮肤护理液的制备方法,其特征在在于,该方法包括以下步骤:
(1)培养脐带间充质干细胞;
(2)从脐带间充质干细胞培养物中提取胞外囊泡浓缩液A,4℃保存;
(3)配制基底液B;
(4)在使用时,将所述胞外囊泡浓缩液A作为皮肤护理液或与基底液B混合搭配为皮肤护理液。
3.如权利要求2所述的方法,其特征在于,在步骤(1)中,所述脐带间充质干细胞的培养过程为:
采集足月剖宫产新生儿脐带剪成2~3cm小段,清洗后剥剪至1mm2小块,均匀铺于培养瓶中,置于37℃、5%CO2浓度的培养箱中培养,8~24h内加入15mL完全培养基覆盖组织块,培养至第五天半换液,此后每隔2~3天换液;
细胞融合度达到70~80%时,吸出组织块加到一个新的培养瓶中,补加培养基到15mL,1~2天后观察新的细胞从组织块周围爬出,融合度达到70%~80%时进行传代,记为P1;
持续传代操作,获得二次贴壁细胞的P2代到P5代作为备用的脐带间充质干细胞。
4.如权利要求2所述的方法,其特征在于,步骤(2)中的胞外囊泡浓缩液A以及步骤(3)基底液B中的干细胞提取物的制备包括以下步骤:
将生长状态良好的5×106脐带间充质干细胞接种至细胞培养皿中,加入无血清的商品化干细胞培养基,细胞的融合度达到80%~90%时,收集培养上清液1;
将上清液1在4℃、以2000g离心8~10min去除完整细胞和死细胞碎片,获得上清液2;
将上清液2在4℃、以10000g离心10min,用0.22μm滤器过滤,得到滤出液1;
将滤出液1使用100kD超滤柱进行浓缩,得到含干细胞外囊泡浓度为1×109~10/mL的胞外囊泡浓缩液A,同时获得非浓缩状态的滤出液2;
将滤出液2使用10kD超滤柱再次进行浓缩,得到干细胞提取物。
5.权利要求1所述的皮肤护理液在制备修复妊娠纹的护肤品中的应用。
6.权利要求1所述的皮肤护理液在制备皮肤II度烧伤及疤痕修复药物中的应用。
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