CN111494297A - 一种含有人脐带间充质干细胞培养上清的精华液 - Google Patents
一种含有人脐带间充质干细胞培养上清的精华液 Download PDFInfo
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Abstract
本发明提供一种人脐带间充质干细胞培养上清，是使用添加了1～10％的血小板裂解物的α‑MEM培养基对人脐带间充质干细胞进行培养，收集P3‑P6代的细胞培养上清制备的。本发明所提供的人脐带间充质干细胞培养上清液用于制备化妆品；所述的化妆品，其一种为精华液。本发明制备的人脐带间充质干细胞培养上清包含多种人体细胞因子，可以直接渗入肌肤，作用于组织细胞，促进皮肤干细胞的修复和再生，从根本上改善皮肤老化。
Description
技术领域
本发明属于化妆品制备技术领域，尤其涉及一种含有人脐带间充质干细胞培养上清的精华液。
背景技术
随着生活水平的提高与科技的进步，以往的美白、保湿等基本功能已不能满足人们对化妆品的需求，抗衰老逐渐成为大家关心的方面。在皮肤老化的过程中，活性氧是皮肤老化的重要诱因，且内源性老化和外源性老化是同时作用的。内源性老化主要是随着年龄的增长，氧化及糖化的蛋白质逐渐累积，使得真皮中胶原蛋白和弹性蛋白损失，皮肤变薄，角质形成细胞变性。外源性老化则是因为紫外线损伤、环境损害、发炎等，导致表皮组织的恶化和损伤。
间充质干细胞(Mesenchymal Stem Cells,MSCs)是来源于早期中胚层的成体干细胞，具有高度自我更新能力和多向分化潜能，是造血微环境中的一种重要的细胞成分，可以向多种组织如骨、软骨、肌肉、韧带、肌腱、脂肪及基质细胞增殖分化，而且免疫原性弱，是组织工程立项的种子细胞来源。人脐带间充质干细胞(human umbilical cord mesenchymalstem cells，hUC-MSCs)相比于其他来源的MSCs，还具有提取方便，含量丰富，细胞纯净，免疫原性更低，细胞原始、分化能力更强，无伦理限制等优势，可为实验和临床提供充足的细胞来源，具有广阔的临床应用前景。研究发现hUC-MSCs的培养上清中含有多种细胞因子，如血管内皮生长因子(vascular endothelial growth factor,VEGF)，肝细胞生长因子(hepatocyte growth factor,HGF)，碱性成纤维细胞生长因子(basic fibroblast growthfactor,b-FGF)，表皮生长因子(epidermal growth factor,EGF)，粒细胞巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)，转化生长因子β(transforming growth factor-β,TGF-β)，神经生长因子(nerve growth factor,NGF)等。而这些因子对于皮肤伤口的愈合及细胞外基质的重建，胶原和弹性蛋白的稳定生产及分布具有重要作用。
目前市场上传统的精华液主要是利用外源性物质，如通过植物成分提取或精细化工制作工艺加工而成的，虽然对肌肤能产生短暂的改善效果，但并未从根本上改变皮肤的衰老问题。尽管现有的专利中有将间充质干细胞培养上清应用于化妆品中，但仅仅是对干细胞进行常规培养，收集培养上清，冷冻干燥或者直接用于化妆品中，含有的细胞因子含量较少，需要额外添加VEGF等生长因子，且生长因子的稳定性并不能保证。
发明内容
有鉴于此，本发明的目的是提供了一种含有人脐带间充质干细胞培养上清的精华液，促进皮肤损伤修复以及抗氧化，且不需要额外添加生长因子，可稳定保存所含生长因子。
本发明首先提供一种人脐带间充质干细胞培养上清，是使用添加了1～10％的血小板裂解物的α-MEM培养基对人脐带间充质干细胞进行培养，收集P3-P6代的细胞培养上清制备的。
其中血小板裂解物的添加浓度为5％。
本发明所提供的人脐带间充质干细胞培养上清液用于制备化妆品；
所述的化妆品，其一种为精华液；
本发明另一个方面还提供一种化妆品精华液，其中使用上述的人脐带间充质干细胞培养上清液；
所述的精华液，其中还添加有鼠尾藻多糖；
所述鼠尾藻多糖的添加质量体积比为1～10％；
所述的精华液，其中还添加有海藻糖；
所述海藻糖的添加质量体积百分比浓度为0.1～5％。
所述的精华液中还添加有化妆品领域使用的保湿剂，其一种具体的保湿剂是甘油；
所述的精华液中还添加有化妆品领域使用的防腐剂，其一种具体的是1,2-戊二醇；
所述的精华液，其一种具体的组成如下：人脐带间充质干细胞培养上清液占50％，鼠尾藻多糖2％，海藻糖0.5％，甘油5％，1,2-戊二醇3％。
与现有技术相比，采用以上技术方案配制的含有人脐带间充质干细胞培养上清的精华液，具有以下优势：
1.本发明制备的人脐带间充质干细胞培养上清包含多种人体细胞因子，可以直接渗入肌肤，作用于组织细胞，促进皮肤干细胞的修复和再生，从根本上改善皮肤老化；
2.使用筛选的MSC无血清培养基，比普通的MSC培养基培养的UC-MSC细胞上清液含有的VEGF、血小板衍生生长因子(Platelet derived growth factor，PDGF)以及b-FGF三种细胞因子多，不需要额外在化妆品中添加，生产成本降低；
3.添加的水溶性鼠尾藻多糖采用超声波破壁结合高压水提技术从鼠尾藻中提取的，具有较强生理活性，能促进人粒细胞超氧阴离子自由基的释放，提高超氧化物歧化酶活性，起到抗氧化作用，为首次应用于化妆品中；
4.通过添加海藻糖及低温冷藏技术有效地保护UC-MSC细胞上清液中细胞因子的活性，从而维持本发明产品的稳定性。
附图说明
图1为40倍显微镜下A、B培养基培养的第五代人脐带间充质干细胞的形态；
图2为A、B培养基培养的第五代人脐带间充质干细胞表面标记物检测；
图3为A、B两种培养基培养的人脐带间充质干细胞培养上清中VEGF、PDGF及b-FGF三种生长因子分泌量的比较；
图4为四组小鼠试验4周后皮肤外观图。
具体实施方式
本发明所用材料来源描述如下：
人脐带间充质干细胞：购于赛业生物；
血小板裂解物：购于美国Compass Biomedical；
鼠尾藻干粉：中国科学院海洋研究所提供。
所检测的VEGF、PDGF及b-FGF三种生长因子在本发明产品中的作用：
VEGF：促进血管内皮细胞增殖，提高局部血管通透性，改善皮肤的新陈代谢；
PDGF：有助于活化伤口愈合，促进新皮肤生长，减少疤痕组织和形成较牢固的血管；
b-FGF：可促进细胞分裂，进行组织修复，促进弹性纤维和胶原蛋白的合成，使肌肤富有弹性。
下面结合附图和实施例对本发明做进一步详细说明。
实施例1制备人脐带间充质干细胞培养上清
一、MSC无血清培养基的筛选
将购买的P2代人脐带间充质干细胞(赛业生物)进行复苏，分别用α-MEM+5％血小板裂解物(A培养基)和纯化学配方(B培养基，具体组成为：谷氨酰胺1-20mM、HEPEs 1-20mM、腐胺10-100μM、转铁蛋白0.1-10μM、维生素C10-400μM、重组胰岛素1-10μM、黄体酮1-20nM、皮质醇10-200nM、人血白蛋白1-20mg/mL、碱性成纤维细胞生长因子1-10ng/mL、转化生长因子β11-10ng/mL、螺旋藻速溶蛋白多糖1-50mg/mL)的两种MSC无血清培养基进行培养。
待细胞长至85％汇合度后进行1:3传代培养，使用0.25％胰蛋白酶消化，收集P3-P6代的细胞培养上清，放于4℃暂时保存。图1为40倍显微镜下两组培养基培养的第五代人脐带间充质干细胞的形态。图2为两组培养基培养的第五代人脐带间充质干细胞表面标记物检测。采用ELISA检测方法对A、B两种培养基培养的UC-MSCs的P2到P10传代细胞的细胞培养上清进行VEGF、PDGF及b-FGF三种生长因子分泌的检测比较，通过制定的标准曲线，计算得到分泌的因子的量。图3为A、B两种培养基培养的人脐带间充质干细胞培养上清中三种生长因子分泌量的比较。
通过对人脐带间充质干细胞的形态、传代，以及VEGF、PDGF和b-FGF三种生长因子的含量检测，综合筛选出适合生产本发明产品的细胞培养基。
结果发现A、B两种培养基培养的P3-P6代人脐带间充质干细胞形态无明显不同，但是B培养基比A培养基贴的更牢固，A培养基只需要用0.125％的胰蛋白酶消化3min即可；而接种相同数量的人脐带间充质干细胞A培养基的增殖数量与B培养基相比差异不显著。根据ELISA检测结果A培养基比B培养基培养的人脐带间充质干细胞的上清中VEGF、PDGF及b-FGF三种生长因子的分泌量显著多。故选定A培养基即α-MEM+5％血小板裂解物为本发明所使用的培养基。
实施例2鼠尾藻多糖的提取
向100g鼠尾藻干粉中加入1000ml去离子水，混匀，在超声波细胞破碎机中破壁30min，然后将破壁后的溶液置于121℃高温高压40min。将高温之后的溶液冷却至室温后，分装到50ml离心管中，10000rpm室温离心30min，收集上清，进行冷冻干燥，获得鼠尾藻多糖干粉。
实施例3含有人脐带间充质干细胞培养上清的精华液混合液的制备
在百级超净台内，将鼠尾藻多糖、海藻糖及1,2-戊二醇用超纯水溶解配置成一定浓度的浓缩液，与干细胞上清超滤浓缩液混合，并加入甘油，用超纯水补足体积至100ml，过0.22μm滤膜过滤除菌。
最终鼠尾藻多糖占总体积的2％(质量体积比)，海藻糖占总体积的0.5％(质量体积比)，甘油占总体积5％，1,2-戊二醇占总体积3％，所述干细胞上清液占总体积50％。将制备的精华液混合液保存于4-8℃下。
实施例4皮肤修复试验
将实验无毛小鼠(7-8周龄，雌性，体重20±5g，共20只)随机分为4组，分别为空白组、模型组、精华液组(实施例3的)和无干细胞上清组(其他成分与实施例3一致，只是人脐带间充质干细胞培养上清由超纯水替代)。模型组、精华液组及无干细胞上清组的小鼠先进行UV灯泡辐射：连续辐照6周，总辐照剂量为4000J/cm2，直至三组小鼠背后均出现光老化皮肤外观，如皮肤增厚、松弛、有皱纹等，若是中间有某只小鼠背部出现红斑、水泡等，则停止辐照，直至症状消失再继续照射。空白组则为是正常小鼠，不做任何处理。给药途径：对精华液组和无干细胞上清组分别进行各自产品涂抹，每日两次，每次20μl/cm2，用手指轻柔均匀涂抹于小鼠背部皮肤，直至其完全吸收，持续给药4周。空白组和模型组不做任何给药。结果见图4。结果发现模型组小鼠皮肤粗糙、红肿，皱纹较深，皮革样外观等；精华液组小鼠皮肤恢复弹性，褶皱减少，外观基本接近空白组小鼠；无干细胞上清组小鼠皮肤粗糙，有皱纹但不红肿。综合说明，本发明产品中的干细胞上清具有修复皮肤损伤的作用。
实施例5精华液抗氧化性检测
碘伏消毒液中的碘具有强氧化性，遇还原性的物质会发生氧化还原反应，使碘伏消毒液由有色变为无色。本实验中将市售的碘伏消毒液稀释100倍，得到棕色的碘伏稀释液。将本发明产品(试验组1)及不含鼠尾藻多糖的溶液(其他成分与本发明一样，鼠尾藻多糖体积由超纯水替代，试验组2)分别缓慢滴加至碘伏稀释液中，边滴加边摇匀，直至碘伏稀释液变为无色，停止滴加，记录两组使用的体积。使用的体积越小，表示产品的还原能力越强。结果见表1，发现试验组1的用量比试验组2显著少，这说明本发明产品的抗氧化功效较好。
表1：碘伏稀释液的氧化还原试验结果表
| 组别 | 试验组1 | 试验组2 |
| 溶液使用量/ml | 0.5 | 2.3 |
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。
Claims (10)
1.一种人脐带间充质干细胞培养上清，其特征在于，所述的培养上清是使用添加1～10％的血小板裂解物的α-MEM培养基对人脐带间充质干细胞进行培养，收集P3-P6代的细胞培养上清制备的。
2.如权利要求1所述的培养上清，其特征在于，所述的血小板裂解物的添加浓度为5％。
3.权利要求1或2所述的培养上清在制备化妆品中的应用。
4.如权利要求3所述的应用，其特征在于，所述的化妆品为精华液。
5.一种化妆品精华液，其特征在于，所述的化妆品精华液中使用权利要求1或2所述的培养上清液。
6.如权利要求5所述的化妆品精华液，其特征在于，所述的化妆品精华液中添加有鼠尾藻多糖。
7.如权利要求6所述的化妆品精华液，其特征在于，所述的鼠尾藻多糖的添加质量体积浓度为1～10％。
8.如权利要求6或7所述的化妆品精华液，其特征在于，所述的精华液中添加有海藻糖。
9.如权利要求6-8任一项所述的化妆品精华液，其特征在于，所述的精华液中添加有化妆品领域使用的保湿剂和/或防腐剂。
10.如权利要求5所述的化妆品精华液，其特征在于，所述的精华液，其一种具体的组成如下：人脐带间充质干细胞培养上清液占50％，鼠尾藻多糖2％，海藻糖0.5％，甘油5％，1,2-戊二醇3％。
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