CN117925513A - 一种脐带间充质干细胞提取方法及应用 - Google Patents
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Abstract
本发明涉及生物医学技术领域，特别涉及一种脐带间充质干细胞提取方法及应用，包括采集符合采集标准的脐带标本，并进行预处理得到脐带组织碎块；将脐带组织碎块进行间充质干细胞分离；将脐带间充质干细胞接种于无血清培养基培养。本发明显著提高了间充质干细胞的扩增效率，而且使得间充质干细胞具有良好的细胞免疫学表型，并结合无血清培养基培养，克服了动物源性血清污染的缺陷和并解决了细胞扩增数量和维持干细胞特性的问题，应用于霜剂化妆品中能显著改善皮肤老化、降低不良反应的发生，增加角质层含水量和油脂含量。
Description
技术领域
本发明涉及生物医学技术领域，特别涉及一种脐带间充质干细胞提取方法及应用。
背景技术
间充质干细胞(Mesenchymal stem cells，MSCs)是一类具有多向分化潜能的成体干细胞，这种干细胞可以自我复制，自我更新，刺激组织生长和修复，并且污染源低，获得方便，生物性能稳定、免疫源性更原始。在适宜的体外实验条件下，容易扩增和保存。间充质干细胞通过改变T淋巴细胞、B淋巴细胞、自然杀伤细胞和树突样细胞分泌的细胞因子，使其体内的免疫细胞增殖，所以间充质干细胞在细胞治疗领域有着极为广阔的应用前景。
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。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和修复问题。
发明内容
针对以上述背景技术的不足，本发明提供一种脐带间充质干细胞提取方法及应用。
一方面，本发明采用的技术方案如下：一种脐带间充质干细胞提取方法，关键在于在于，包括以下步骤：
S1.采集符合采集标准的脐带标本，并进行预处理得到脐带组织碎块；
S2.将脐带组织碎块进行间充质干细胞分离；
S3.将脐带间充质干细胞接种于无血清培养基培养；
所述无血清培养基包括DMEM-F12培养基、血小板源性因子1-50ng/mL，较佳的为1-20ng/mL、碱性成纤维生长因子20-50ng/mL，较佳的为25-40ng/mL、芦荟抽提物10-50ng/mL，较佳的为30-40ng/mL、人参抽提物10-50ng/mL，较佳的为10-20ng/mL、肉苁蓉抽提物10-50ng/mL，较佳的为30-40ng/mL。
优选的，所述S1具体为从保存液中取出脐带，物理破碎脐带，去除脐带结扎两端外侧，中间段剪成1～2cm长的小段，用生理盐水反复冲洗脐带小段，剖开，去除其动脉、静脉，用生理盐水反复冲洗去除血液和血块，用10倍双抗浸泡后用剪刀剪碎至0.5～2mm3，得到脐带组织碎块。
优选的，所述S2具体为将脐带组织碎块采用I型胶原酶溶液、EDTA溶液、胰蛋白酶的顺序消化，每次消化的条件为：温度36～38℃，时间20～30min，终止消化后用100um网筛过滤组织残渣，收集含细胞的滤液，洗涤离心得到的间充质干细胞。
优选的，所述S3的培养条件为温度为37℃，二氧化碳体积比为5-7％、氧气的体积比为5-7％。
优选的，间充质干细胞数量达到80％～90％融合度时，按1:3比例传代培养。
另一方面，本发明提供一种脐带血间充质干细胞的在化妆品中的应用。
另一方面，本发明提供一种抗衰老美容产品，包括质量分数为0.5-20％的脐带血间充质干细胞。
有益效果：与现有技术相比，本发明采用物理破碎联合消化酶的方法从脐带提取间充质干细胞，该扩增方法显著提高了间充质干细胞的扩增效率，而且使得间充质干细胞具有良好的细胞免疫学表型，并结合无血清培养基培养，克服了动物源性血清污染的缺陷和并解决了细胞扩增数量和维持干细胞特性的问题，不含血清，避免了不明血清成份对细胞培养的影响和动物源性的污染，促进细胞增殖并维持干细胞特性，增加了人脐带间充质干细胞的存活率，应用于霜剂化妆品中能显著改善皮肤老化、降低不良反应的发生，增加角质层含水量和油脂含量。
具体实施方式
为使本领域技术人员更好的理解本发明的技术方案，下面结合具体实施方式对本发明作详细说明。在以下实施例中所涉及的仪器设备如无特别说明，均为常规仪器设备；所涉及的工业原料(试剂、原料视情况选择)如无特别说明，均为市售常规工业原料；所涉及的加工制作方法(检测、测试、制备方法等视情况而选择)，如无特别说明，均为常规方法。
实施例1脐带间充质干细胞提取方法
从保存液中取出脐带，物理破碎脐带，去除脐带结扎两端外侧，中间段剪成1～2cm长的小段，用生理盐水反复冲洗脐带小段，剖开，去除其动脉、静脉，用生理盐水反复冲洗去除血液和血块，用10倍双抗浸泡后用剪刀剪碎至0.5～ 2mm3，得到脐带组织碎块；将脐带组织碎块采用I型胶原酶溶液、EDTA溶液、胰蛋白酶的顺序消化，每次消化的条件为：温度36～38℃，时间20～30min，终止消化后，用生理盐水洗两次，用100um网筛过滤组织残渣，收集含细胞的滤液，洗涤离心得到的间充质干细胞；将脐带间充质干细胞接种于无血清培养基，所述无血清培养基包括DMEM-F12培养基、血小板源性因子15ng/mL、碱性成纤维生长因子35ng/mL、芦荟抽提物35ng/mL、人参抽提物10ng/mL、肉苁蓉抽提物40ng/mL，在温度为37℃，二氧化碳体积比为5％、氧气的体积比为 6％的培养箱中孵育15-25h，更换新的培养基，待间充质干细胞数量达到85％融合度时，按1:3比例传代培养，各代培养所用的脂肪间质干细胞的细胞密度为 2000个细胞/cm2，检测结果如下表1所示。
| 代数 | 细胞存活数量 | 增加倍数 | 存活率 |
| P1 | 2.47×106 | 117.8 | 96.7％ |
| P2 | 3.24×106 | 154.6 | 97.1 |
| P3 | 1.78×106 | 84.9 | 95.8 |
实施例2含有脐带间充质干细胞提取物的美容面霜
所述的美容面霜包括以下组分及其质量百分比，实施例2制得的间充质干细胞提取物8.5％、羊毛醇3.5％、白蜂蜡7.5％、山茶花提取物1.3％、谷胱甘肽5％、肌肽2％、余量为水。
试验方法：筛选60例皮肤出现老化的女性受试志愿者，年龄30-50岁，平均年龄40岁，入选标准：皮肤干燥、弹性降低、出现皱纹、色素沉着、肤色灰暗等皮肤老化现象，在整个试验期间不使用其他抗衰老产品。志愿者分为实验组和对照组，每组30例，实验组使用为受试者使用实施例2产品，对照组为受试者不使用任何护肤产品，早晚洁面后取适量的美容面霜涂抹于面部，各组整个试验时间为60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。安全性评估：主要评价使用产品后受试者出现轻微不适、红斑、干燥、瘙痒和刺痛等不良反应的发生情况，试验结果如表2所示。
表2
1-轻微不适，2-红斑，3-干燥，4-瘙痒，5-刺痛含水量、油脂含量评估：分别在0天、60天及100天对受试者采用无创性仪器检测受试部位的含水量、油脂含量，进行统计分析。试验结果如表3所示。
表3
最后需要说明，上述描述仅为本发明的优选实施例，本领域的技术人员在本发明的启示下，在不违背本发明宗旨及权利要求的前提下，可以做出多种类似的表示，这样的变换均落入本发明的保护范围之内。
Claims (8)
1.一种脐带间充质干细胞提取方法，其特征在于，包括以下步骤：
S1.采集符合采集标准的脐带标本，并进行预处理得到脐带组织碎块；
S2.将脐带组织碎块进行间充质干细胞分离；
S3.将脐带间充质干细胞接种于无血清培养基培养；
所述无血清培养基包括DMEM-F12培养基、血小板源性因子1-50ng/mL；碱性成纤维生长因子20-50ng/mL；芦荟抽提物10-50ng/mL；人参抽提物，10-50ng/mL；肉苁蓉抽提物，10-50ng/mL。
2.根据权利要求1所述的一种脐带间充质干细胞提取方法，其特征在于所述S1具体为从保存液中取出脐带，物理破碎脐带，去除脐带结扎两端外侧，中间段剪成1～2cm长的小段，用生理盐水反复冲洗脐带小段，剖开，去除其动脉、静脉，用生理盐水反复冲洗去除血液和血块，用10倍双抗浸泡后用剪刀剪碎至0.5～2mm3，得到脐带组织碎块。
3.根据权利要求1所述的一种脐带间充质干细胞提取方法，其特征在于所述S2具体为将脐带组织碎块采用I型胶原酶溶液、EDTA溶液、胰蛋白酶的顺序消化，每次消化的条件为：温度36～38℃，时间20～30min，终止消化后用100um网筛过滤组织残渣，收集含细胞的滤液，洗涤离心得到的间充质干细胞。
4.根据权利要求2所述的一种脐带间充质干细胞提取方法，其特征在于所述S3的培养条件为温度为37℃，二氧化碳体积比为5-7％、氧气的体积比为5-7％。
5.根据权利要求2所述的一种脐带间充质干细胞提取方法，其特征在于间充质干细胞数量达到80％～90％融合度时，按1:3比例传代培养。
6.权利要求1-5任一项所述的方法提取的脐带血间充质干细胞在化妆品中的应用。
7.一种抗衰老美容产品，其特征在于：包括权利要求1-5任一项所述的方法提取的脐带血间充质干细胞。
8.根据权利要求7所述的一种抗衰老美容产品，其特征在于：所述脐带血间充质干细胞的质量分数为0.5-20％。
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