CN113499306A - 一种脐带间充质干细胞中高分子提取物、应用及检测方法 - Google Patents
一种脐带间充质干细胞中高分子提取物、应用及检测方法 Download PDFInfo
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Abstract
本发明提供一种脐带间充质干细胞中高分子提取物、应用及检测方法,化妆品技术领域,其中脐带间充质干细胞中高分子提取物,包括下述步骤:(1)从脐带组织块或脐血中分离得到脐带间充质干细胞;(2)将步骤(1)得到的脐带间充质干细胞定向分化为皮肤干细胞;(3)将步骤(2)得到的皮肤干细胞收集和裂解,并通过孔径为4~20.0nm分子筛将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物;本发明的这类中高分子物质活性物质的浓度高、对皮肤具有抗敏、修复能力强。
Description
技术领域
本发明涉及化妆品技术领域,尤其涉及一种脐带间充质干细胞中高分子提取物、应用及检测方法。
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。皮肤的老化是一个持续渐进的生理过程。在外观上表现为皮肤干燥、松弛、皱纹、色素的沉积,老化的发生包括多方面因素;皮肤的衰老主要分为自然老化和光老化。自然老化就是由机体内在因素的作用所引起,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,常见于暴露部位和非暴露部位,明显特征为皱纹的出现和皮肤的松弛。光老化则是指皮肤衰老受紫外辐照诱导的损伤和内源性损伤的叠加,表现为皮肤暴露部位粗糙、表皮角质损伤、皮肤弹性胶原蛋白减少、金属蛋白酶类的增加、炎症浸润和血管扩张。
间充质干细胞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相比于其他来源的间充质干细胞,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。研究发现人脐带间充质干细胞经裂解后中含有多种细胞因子,碱性成纤维细胞生长因子,表皮生长因子,粒细胞巨噬细胞集落刺激因子,转化生长因子β,神经生长因子等。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和修复问题。申请号为CN201910177644.0,发明名称为一种用于常规抗衰老治疗的人源脐带间充质干细胞制剂的制备方法公开了包括脐带组织块的获取步骤、干细胞的原代培养步骤、干细胞的传代培养步骤、干细胞的冻存步骤、干细胞的复苏与培养步骤和人源脐带间充质干细胞制剂的制备步骤;所述人源脐带间充质干细胞制剂的制备步骤包括采用5%葡萄糖注射液重悬传代培养至Pe代人源脐带间充质干细胞,该发明完善人源脐带间充质干细胞的储备,保障了人源脐带间充质干细胞的存活率、增殖能力和特异性。但是,现有技术使用的间充质干细胞本身以及其分泌的旁分泌因子或者外泌体活性物质的浓度低、生产成本高。
发明内容
有鉴于此,本发明提供一种脐带间充质干细胞中高分子提取物,以解决现行技术中脐带间充质干细胞中高分子提取物活性成分含量低、抗敏、修复能力弱。
为解决上述技术问题,本发明提供一种脐带间充质干细胞中高分子提取物,包括下述步骤:(1)从脐带组织块或脐血中分离得到脐带间充质干细胞;(2)将步骤(1)得到的脐带间充质干细胞定向分化为皮肤干细胞;(3)将步骤(2)得到的皮肤干细胞收集和裂解,并通过孔径为4~20.0nm分子筛将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。
优选的,其由下述步骤提取得到:(1)从脐带组织块中分离得到脐带间充质干细胞;(2)将步骤(1)得到的脐带间充质干细胞定向分化为皮肤干细胞;(3)培养皮肤干细胞,并在对数期采用50~100mJ/cm2紫外辐照细胞6~8h;(4)将步骤(3)得到皮肤干细胞正常培养18~36h后,再置于湿度为80~85%、氧气6~8%、二氧化碳5~6%、余量为氮气的低氧环境下培养18~36h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。
优选的,步骤(1)中,所述脐带间充质干细胞是从脐带组织块或脐血中经过原代培养步骤、干细胞的传代培养步骤、干细胞的冻存步骤、干细胞的复苏与培养步骤和人源脐带间充质干细胞制剂的制备步骤得到的。
优选的,在步骤(2)中,所述的间充质干细胞培养基为无血清培养基:所述无血清培养基是在无血清基础培养基中添加终浓度为4.5wt%~5.5wt%的UltraGRO无血清培养基、45μg/ml~55μg/ml的维生素C、15ng/ml~25ng/ml的干细胞生长因子、1.5mmol/ml~2.5mmol/ml的L-谷氨酰胺、90ng/ml~110ng/ml的人转化生长因子TGF-β1、15ng/ml~25ng/ml的表皮生长因子和15ng/ml~25ng/ml的人纤维细胞生长因子、黄芩甙元0.1~0.3mg/L。
优选的,在步骤(4)中,所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300~350W,每工作10s,间歇3s,循环至少20次。
优选的,一种脐带间充质干细胞中高分子提取物的抗敏修复化妆品,其特征在于:包括如下百分含量的原料组成:脐带间充质干细胞中高分子提取物13~18%、牛蒡子浓缩提取液3~5%、玉兰提取液5~8%、茶多酚1~3%、仙人掌萃取液3~5%、玻璃苣籽油1~3%、银耳多糖0.3~0.5%、SOD 2~3%、肌肽1~3%、L-苹果酸3~4%、蜂胶3%、余量为水。
优选的,所述牛蒡子浓缩提取液的制备方法包括如下步骤:将牛蒡子粉碎过40目筛网,采用并放入采用逆流浸提柱法提取牛蒡子中有效成分,得到牛蒡子提取液A,并取出牛蒡子渣加入质量浓度为70~80%的酒精,按1:10~15的固液质量比在75~85℃下提取3次,合并得到提取液;提取液经减压蒸干至恒重,得到牛蒡子浓缩提取物。
优选的,所述玉兰提取液的制备方法包括如下步骤:取新鲜玉兰叶洗净、沥干,通过粉碎机将玉兰叶打至成糊状,按1g叶浆加3~5mL体积浓度为75%的乙醇并进行间歇的超声震荡,每超声震荡3min,停歇1min,重复4~6次。
优选的,所述仙人掌萃取液的制备方法如下:仙人掌打成浆液,按照100g浆液加入乳酸菌活菌105CFU和酵母菌104CFU,发酵48~72h,加入75%的乙醇并进行间歇的超声震荡,加入有机溶剂进行萃取,最后旋蒸仪得到仙人掌萃取液。
一种脐带间充质干细胞中高分子提取物的检测方法,采用反相液质联用方法。
与现有技术相比,本发明的有益技术效果在于:
1.本发明先把间充质干细胞定向分化为皮肤干细胞,再把皮肤干细胞收集起来,将得到的皮肤干细胞收集和裂解,再过分子筛富集中高分子物质。这类中高分子物质对于抗敏修复方面能力明显提升。
2.本发明优选的在细胞的培养的对数期对细胞采用低紫外辐照的方式,诱导细胞抗皮肤紫外损伤物质的产生数量,同时对细胞起到筛选的作用。同时将经过紫外处理的细胞经过正常培养,使细胞恢复正常增值和生长,使细胞达到足够的数量,再至于湿度为80~85%、氧气6~8%、二氧化碳5~6%、余量为氮气的低氧环境下培养18~36h后,此环境相对正常培养,为低氧低湿环境,细胞在相对波动环境,会产生大量生长因子维持其生长的状态,从而提高了提取物的产量和活性物质的含量。
而与现有技术中直接使用间充质干细胞本身以及其分泌的旁分泌因子或者外泌体,并不进行过滤处理,其中的大分子对皮肤屏障修复能力差,常温下其中的活性物质容易失活,并且不能常温保存。我们通过把间充质干细胞定向分化为皮肤干细胞,再富集其中高分子活性物质,使其在常温也能保存比较长的时间,并且增强对皮肤屏障的修复能力。
3.本发明的抗敏修复化妆品,脐带间充质干细胞中高分子提取物与牛蒡子浓缩提取液和玉兰提取液相配合,针对皮肤因紫外辐射造成的辐射效果显著。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。
附图说明
图1为实施例1-6及对比例1-2的细胞提取物对细胞指数的图;
图2为实施例1-6及对比例1-2细胞提取物对成纤维细胞胶原蛋白生成量的图;
图3为实施例1、实施例7-12细胞提取物对成纤维细胞胶原蛋白生成量的图;
图4为实施例11、实施例13-15的细胞提取物对细胞指数的图;
图5为实施例11、实施例13-15细胞提取物对成纤维细胞胶原蛋白生成量的图;
图6为实施例14、实施例16-20的细胞提取物对细胞指数的图;
图7为实施例14、实施例16-20细胞提取物对成纤维细胞胶原蛋白生成量的图;
图8为实施例17、实施例21-22细胞提取物对成纤维细胞胶原蛋白生成量的图;
图9为实施例17及对比例3-4的细胞提取物对细胞指数的图;
图10为实施例17及对比例3-4细胞提取物对成纤维细胞胶原蛋白生成量的图;
图11为200ngHeLa液质分析基峰色谱图;
图12为母离子误差分布图;
图13为细胞样品液质分析基峰色谱图;
图14为细胞样品母离子误差分布图;
图15细胞样品肽段漏切率分布;
图16细胞培养基样品液质分析BasePeak图;
图17细胞样品肽段漏切率分布;
图18为使用本发明的脐带间充质干细胞中高分子提取物对于患有红肿过敏患者的影响。
具体实施方式
下面结合附图和实施例来说明本发明的具体实施方式,但以下实施例只是用来详细说明本发明,并不以任何方式限制本发明的范围。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1:一种脐带间充质干细胞中高分子提取物,其由下述步骤提取得到:
(1)从脐带组织块中分离得到脐带间充质干细胞,采集符合采集标准的脐带标本,于48h内进行细胞分离。将脐带保存液弃尽,用PBS冲洗3次,并将脐带移至Ф10cm培养皿内,截取透亮度好、无破损、无水肿区域的脐带小段,每段截成4*4cm。用组织剪将脐带纵向剖开,剔除脐静脉和脐动脉,用PBS冲洗4次。使用脐带组织专用剪将脐带剩余组织即华通氏胶剪成1mm3的组织块,接种至新的Ф10cm培养皿内,24h后补一半无血清完全培养基,后每2d进行弃3ml补4ml的半量换液操作;
(2)将步骤(1)得到的脐带间充质干细胞定向分化为皮肤干细胞;在步骤(2)中,所述的间充质干细胞培养基为无血清培养基:所述无血清培养基是在无血清基础培养基中添加终浓度为5wt%的UltraGRO无血清培养基、50μg/ml的维生素C、20ng/ml的干细胞生长因子、2mmol/ml的L-谷氨酰胺、100ng/ml的人转化生长因子TGF-β1、20ng/ml的表皮生长因子和20ng/ml的人纤维细胞生长因子、黄芩甙元0.2mg/L;
(3)培养皮肤干细胞,并在对数期采用70mJ/cm2紫外辐照细胞7h;
(4)将步骤(3)得到皮肤干细胞正常培养24h后,再置于湿度为82%、氧气7%、二氧化碳5%、余量为氮气的低氧环境下培养24h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
实施例2:与实施例1的不同之处在于步骤(3):
(3)培养皮肤干细胞,并在对数期采用50mJ/cm2紫外辐照细胞7h;
实施例3:与实施例1的不同之处在于步骤(3):
(3)培养皮肤干细胞,并在对数期采用60mJ/cm2紫外辐照细胞7h;
实施例4:与实施例1的不同之处在于步骤(3):
(3)培养皮肤干细胞,并在对数期采用80mJ/cm2紫外辐照细胞7h;
实施例5:与实施例1的不同之处在于步骤(3):
(3)培养皮肤干细胞,并在对期采用90mJ/cm2紫外辐照细胞7h;
实施例6:与实施例1的不同之处在于步骤(3):
(3)培养皮肤干细胞,并在对期采用100mJ/cm2紫外辐照细胞7h;
对比例1:与实施例1的不同之处在于步骤(3):
(3)培养皮肤干细胞,并在对期采用110mJ/cm2紫外辐照细胞7h;
对比例2:与实施例1的不同之处在于步骤(3):
(3)培养皮肤干细胞,并在对期采用40mJ/cm2紫外辐照细胞7h;
实施例1-6及对比例1-2,在细胞培养过程中,经过紫外照射后测定其培养液中细胞指数,如图1所示。
收集实施例1-6及对比例1-2中的提取物加入至成纤维细胞培养48h后,将成纤维细胞进行裂解,取各组细胞上清,加入100uL胶原分离与浓缩试剂,4℃过夜后离心弃上清。将标准胶原分别稀释为0、0.01、0.05、0.10、0.20、0.5、1.00mg·mL-1。各样本管中加入500μLsircol染料孵育30min,用1×Acid-SaltWashReagent清洗。向各管中加入250uL碱性金属试剂,涡旋混匀。转移200μL样本至96孔板中。用酶标仪检测,检测波长为555nm,用水调吸光值为零,测定空白试剂、标准胶原和检测样本空白试剂吸光度,读数重复试验误差士10%,重复3次,实施例1、实施例3、实施例4的胶原蛋白的检测结果如图2所示。
由图1和2可知,培养皮肤干细胞,在对期采用70mJ/cm2紫外辐照细胞得到细胞数虽然低于不加紫外辐照和50mJ/cm2及60mJ/cm2,但是其提取液作用于纤维细胞培养上对胶原蛋白的生产量上显著高于其他组,由图1可知,采用紫外灯高于110mJ/cm2,会造成大量的细胞死亡,最终得到的提取物对于成纤维细胞胶原蛋白产生量也大大减少。
同时研究发现,辐照时间过长会造成细胞一段时间后不在增长;时间过短,起不到筛选的作用。
实施例7:与实施例1的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养18h后,再置于湿度为82%、氧气7%、二氧化碳5%、余量为氮气的低氧环境下培养24h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
实施例8:与实施例1的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养30h后,再置于湿度为82%、氧气7%、二氧化碳5%、余量为氮气的低氧环境下培养24h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
实施例9:与实施例1的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养24h后,再置于湿度为82%、氧气7%、二氧化碳5%、余量为氮气的低氧环境下培养18h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
实施例10:与实施例1的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养24h后,再置于湿度为82%、氧气7%、二氧化碳5%、余量为氮气的低氧环境下培养21h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
实施例11:与实施例1的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养24h后,再置于湿度为82%、氧气7%、二氧化碳5%、余量为氮气的低氧环境下培养27h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
实施例12:与实施例1的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养24h后,再置于湿度为82%、氧气7%、二氧化碳5%、余量为氮气的低氧环境下培养30h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
收集实施例1、实施例7-12的提取物加入至成纤维细胞培养48h后,将成纤维细胞进行裂解,取各组细胞上清,加入100uL胶原分离与浓缩试剂,4℃过夜后离心弃上清。将标准胶原分别稀释为0、0.01、0.05、0.10、0.20、0.5、1.00mg·mL-1。各样本管中加入500μLsircol染料孵育30min,用1×Acid-SaltWashReagent清洗。向各管中加入250uL碱性金属试剂,涡旋混匀。转移200μL样本至96孔板中。用酶标仪检测,检测波长为555nm,用水调吸光值为零,测定空白试剂、标准胶原和检测样本空白试剂吸光度,读数重复试验误差士10%,重复3次,实施例1、实施例7-12的胶原蛋白的检测结果如图3所示。
由图3可知,实施例1、实施例10及实施例11中胶原蛋白的含量高于其他实施例组,从节省时间的角度,选用实施例10在低氧环境下培养21h。
实施例13:与实施例11的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养24h后,再置于湿度为80%、氧气7%、二氧化碳5%、余量为氮气的低氧环境下培养27h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
实施例14:与实施例11的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养24h后,再置于湿度为84%、氧气7%、二氧化碳5%、余量为氮气的低氧环境下培养27h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
实施例15:与实施例11的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养24h后,再置于湿度为85%、氧气7%、二氧化碳5%、余量为氮气的低氧环境下培养27h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
实施例11、实施例13-15培养出的此阶段细胞,细胞指数图如图4所示。
收集实施例11、实施例13-15的提取物加入至成纤维细胞培养48h后,将成纤维细胞进行裂解,取各组细胞上清,加入100uL胶原分离与浓缩试剂,4℃过夜后离心弃上清。将标准胶原分别稀释为0、0.01、0.05、0.10、0.20、0.5、1.00mg·mL-1。各样本管中加入500μLsircol染料孵育30min,用1×Acid-SaltWashReagent清洗。向各管中加入250uL碱性金属试剂,涡旋混匀。转移200μL样本至96孔板中。用酶标仪检测,检测波长为555nm,用水调吸光值为零,测定空白试剂、标准胶原和检测样本空白试剂吸光度,读数重复试验误差士10%,重复3次,实施例11、实施例13-15的胶原蛋白的检测结果如图5所示。
由图4、5可知,在湿度为82%~85%,并不影响细胞的数量,就胶原蛋白的产生量上说,实施例14的提取物对成纤维细胞胶原蛋白的产生量最高。
实施例16:与实施例14的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养24h后,再置于湿度为84%、氧气6%、二氧化碳5%、余量为氮气的低氧环境下培养27h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
实施例17:与实施例14的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养24h后,再置于湿度为84%、氧气8%、二氧化碳5%、余量为氮气的低氧环境下培养27h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
实施例18:与实施例14的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养24h后,再置于湿度为84%、氧气6%、二氧化碳6%、余量为氮气的低氧环境下培养27h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
实施例19:与实施例14的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养24h后,再置于湿度为84%、氧气7%、二氧化碳6%、余量为氮气的低氧环境下培养27h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
实施例20:与实施例14的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养24h后,再置于湿度为84%、氧气8%、二氧化碳6%、余量为氮气的低氧环境下培养27h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
实施例14、实施例16-20培养出的此阶段细胞,细胞指数图如图6所示。
收集实施例14、实施例16-20培养出的提取物加入至成纤维细胞培养48h后,将成纤维细胞进行裂解,取各组细胞上清,加入100uL胶原分离与浓缩试剂,4℃过夜后离心弃上清。将标准胶原分别稀释为0、0.01、0.05、0.10、0.20、0.5、1.00mg·mL-1。各样本管中加入500μLsircol染料孵育30min,用1×Acid-SaltWashReagent清洗。向各管中加入250uL碱性金属试剂,涡旋混匀。转移200μL样本至96孔板中。用酶标仪检测,检测波长为555nm,用水调吸光值为零,测定空白试剂、标准胶原和检测样本空白试剂吸光度,读数重复试验误差士10%,重复3次,实施例14、实施例16-20的胶原蛋白的检测结果如图7所示。
由图6、7可知,实施例17环境条件即在氧气8%、二氧化碳5%、余量为氮气的低氧环境下培养的纤维细胞胶原蛋白的产生量最高。
实施例21:与实施例17的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养24h后,再置于湿度为84%、氧气8%、二氧化碳5%、余量为氮气的低氧环境下培养27h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为4.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
实施例22:与实施例17的不同之处在于步骤(4):
(4)将步骤(3)得到皮肤干细胞正常培养24h后,再置于湿度为84%、氧气8%、二氧化碳5%、余量为氮气的低氧环境下培养27h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为20nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300W,每工作10s,间歇3s,20次。
收集实施例17、实施例21-22培养出的提取物加入至成纤维细胞培养48h后,将成纤维细胞进行裂解,取各组细胞上清,加入100uL胶原分离与浓缩试剂,4℃过夜后离心弃上清。将标准胶原分别稀释为0、0.01、0.05、0.10、0.20、0.5、1.00mg·mL-1。各样本管中加入500μLsircol染料孵育30min,用1×Acid-SaltWashReagent清洗。向各管中加入250uL碱性金属试剂,涡旋混匀。转移200μL样本至96孔板中。用酶标仪检测,检测波长为555nm,用水调吸光值为零,测定空白试剂、标准胶原和检测样本空白试剂吸光度,读数重复试验误差士10%,重复3次,实施例17、实施例21-22的胶原蛋白的检测结果如图8所示。
由图8可知,采用10nm和20nm的分子筛,对成纤维细胞产生胶原蛋白的产生量相当。
对比例3:与实施例17的不同之处在于步骤(3):
(3)培养皮肤干细胞,不经过紫外辐照;
对比例4:与实施例17的不同之处在于步骤(3):
(4)将步骤(3)得到皮肤干细胞正常培养48h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。
实施例17及对比例3-4中在细胞裂解前测定其中细胞浓度,测定结果如图9所示。
实施例17及对比例3-4,提取物1天内加入至成纤维细胞培养48h后,将成纤维细胞进行裂解,取各组细胞上清,加入100uL胶原分离与浓缩试剂,4℃过夜后离心弃上清。具体操作如上述。实施例9、实施例11-14、对比例1提取物成纤维细胞生成胶原蛋白的影响如图10所示。
由图10可以看出,经过紫外辐照和低氧处理对对活性物质的产生具有协调的促进作用。
一种脐带间充质干细胞中高分子提取物的检测方法如下,将本发明实施例17的脐带间充质干细胞中高分子提取物做如下检测:
1.检测方法:蛋白质提取→蛋白质处理→C18反相Tip柱除盐→反相液质联用RPLC-MS。
蛋白质提取
(1)细胞样品:向EP管中加入300μL含有1%蛋白酶抑制剂的1%SDS溶液,冰浴超声破碎2min(10son,10off,避免过热损失蛋白质);21000×g4℃离心15min,取上清;采用BCA发测定蛋白质浓度。
(2)培养基样品:取1mL培养基,加入2mL甲醇,涡旋混匀,冰上静置5min;21000×g4℃离心15min,弃上清;加入300μL1%SDS溶液,涡旋溶解蛋白质沉淀;采用BCA发测定蛋白质浓度。
蛋白质处理
(1)上述两种蛋白质溶液相同处理,如下:溶液95℃变性10min,冷却至室温;向离心管中加入3μL1M二硫苏糖醇(DTT),56℃恒温振荡1.5h;冷却至室温,加入6μL1M碘乙酰胺(IAA),避光反应30min。
(2)使用FASP(filter-aidedsamplepreparation)酶解方法进行酶解:200μL50mM碳酸氢铵(ABC)润洗超滤膜,取50μg处理的蛋白质上样至超滤膜上,离心去除缓冲溶液;加入200μL50mMABC溶液,充分将SDS去除;加入100μLpH8缓冲溶液,1μgTrypsin蛋白酶,37℃恒温振荡反应12h;加入5μL三氟乙酸(TFA)终止酶解,更换离心管,离心收集肽段混合物。
反相Tip柱除盐
(1)活化:使用1.5mL离子管(盖子上用尖锐工具打直径0.3cm的孔)固定Pep-Tip除盐柱(仅用于处理50μg样品),加入200μLBPhase,5000×g(或7000rpm)离心5min;
(2)平衡:加入200μLAPhase,5000×g(或7000rpm)离心5min;
(3)吸附样品:加入样品,3000×g(或5000rpm)离心10min;
(4)脱盐:200μLAPhase清洗柱子,5000×g(或7000rpm)离心5min;
(5)洗脱:更换低吸附离心管收集样品,200μLBPhase,5000×g(或7000rpm)离心5min,冻干洗脱液。
(6)溶解:离心管中加入20μLAPhase,-20℃冻存,等待上样。
反相液质联用RPLC-MS
(1)数据采集软件:Xcalibur(Thermo,USA);色谱柱信息:单柱模式,分离柱:75μm×200mm(C18,1.9μm粒径,120A孔径);色谱仪器:Easy-nano1000;质谱仪器:QExactivePlus(Thermo,USA)
(2)色谱条件:分析柱平衡体积:5μL;上样量:3μL;Loading体积:8μL
色谱分离时间:120min;A:0.1%甲酸水溶液;B:80%乙腈,0.1%甲酸
流速:200nL/min;梯度:
(4)质谱条件:MS扫描范围(m/z)355-1700,分辨率70,000,最大离子注入时间100ms,MS/MS离子注入时间75ms,扫描范围200-2000,分辨率35,000,碎裂模式:高能碰撞(HCD),碎裂能量(NCE):27;排除1价及高于7价的离子,动态排除时间60s,数据依赖采集模式(DDA),一个MS1图谱选择10个最强的母离子进行串级扫描。
数据分析
质谱产生的原始数据.Raw文件通过ProteinDiscovery2.2.0(Thermo)进行定性分析,ProteinDiscovery参数如下:数据库为UniProtKB(2015_04,42121),其他参数设置如下:母离子质量容差10ppm;碎片离子质量容差:0.05Da;蛋白质酶:Trypsin;最多允许2个漏切位点;固定修饰:carbamidomethyl(C);可变修饰:oxidation(M)和acetyl(proteinN-term)。
检测结果
2.1质控数据:
样品:200ngHeLa液质分析基峰色谱图;样品量:200ng,检测结果如图11所示。
由图11可知,肽段高效分离是保证质谱检测的前提,图11中,提取离子色谱图峰宽<0.5min,说明肽段得到很好的分离;色谱峰平滑,并未出现明显毛刺,说明电喷雾(ESI)稳定,可以实现肽段的高效离子化。
图12可以发现质谱质量轴偏差小于2ppm,充分说明质谱状态正常;从20ngHeLa酶解产物中鉴定的肽段数目9,505条,蛋白质鉴定数目2439,该数据也说明液质联用***状态良好,可用于样品测定。
蛋白质鉴定列表做以下说明:
coverage:覆盖率;Peptides:所有肽段;PSMs:鉴定次数;UniquePeptides:该蛋白质组的唯一肽段数;AAs:蛋白序列长度;MW:蛋白质的分子量;calc.pI:蛋白质的等电点。K-Q列:每个样本定量情况;R-X列:每个样本的定量值;ScoreMascot:蛋白搜库打分;PeptidesMascot:肽段搜库打分;
肽段鉴定结果的每列做以下说明:
Sequence:肽段序列;Modifications:肽段修饰;Qvalityq-value:实际RDR(假阳性率),值越小越好;ProteinGroups:出现在几个ProteinGroups中;Proteins:出现在几个蛋白中;PSMs:鉴定次数;MasterProteinAccessions:最佳匹配蛋白号;MissedCleavages:漏切位点;Theo.MH+[Da]:理论质荷比;J-P列:肽段在每个样本中的定量情况;Q-中的定量值;IonsScoreMascot:母离子打分;W:肽段在每个样本。
(2)细胞样品鉴定结果
样本信息:人源细胞;上样量:200ng,细胞样品鉴定结果如图13所示,
细胞样品母离子误差分布图如图13所示;
细胞样品肽段漏切率分布如图14所示。
图13、14说明液质联用***可以很好的实现样品处理后肽段的高效分析,质谱状态稳定。
图15显示包含少于2个漏切位点的肽段占比达到99.5%,充分说明样品前处理方法完全可以很好的进行样品预处理,即超声波提取+FASP膜酶解。采用该方法从200ng细胞样品中鉴定的蛋白数目为3609。
(3)细胞培养基样品鉴定结果
样本信息:人源细胞;上样量:200ng。人源细胞培养基样品液质分析BasePeak图如图16所示,细胞样品肽段漏切率分布如图17所示。
图16、17包含少于2个漏切位点的肽段占比为96%,基峰色谱图图显示肽段较少,说明样品主要是高丰度蛋白质。
表2为本发明脐带间充质干细胞中高分子提取物检测结果共3000多种,下表取代表性的一部分。
实施例23
一种含有脐带间充质干细胞中高分子提取物的抗敏修复化妆品,由以下质量百分比组分组成,实施例17制得的间充质干细胞提取物15%、牛蒡子浓缩提取液4%、玉兰提取液6%、茶多酚2%、仙人掌萃取液4%、玻璃苣籽油2.5%、银耳多糖0.4%、SOD2.5%、肌肽2%、L-苹果酸3.6%、蜂胶3%、余量为水。
在本实施例中,牛蒡子浓缩提取液的制备方法包括如下步骤:将牛蒡子粉碎过40目筛网,采用并放入采用逆流浸提柱法提取牛蒡子中有效成分,得到牛蒡子提取液A,并取出牛蒡子渣加入质量浓度为75%的酒精,按1:12的固液质量比在80℃下提取3次,合并得到提取液;提取液经减压蒸干至恒重,得到牛蒡子浓缩提取物。采用此种方式浓缩提取物,得到的牛蒡子浓缩提取物工艺相对简单,可以将牛蒡子内不易提取的物质经过酒精,利用相似相容的原理,将其中的活性物质尽可能的提取出,再利用减压蒸干将牛蒡子浓缩提取物不至于被破坏。
在本实施例中,所述玉兰提取液的制备方法包括如下步骤:取新鲜玉兰叶洗净、沥干,通过粉碎机将玉兰叶打至成糊状,按1g叶浆加4mL体积浓度为75%的乙醇并进行间歇的超声震荡,每超声震荡3min,停歇1min,重复6次。
在本实施例中,所述仙人掌萃取液的制备方法如下:仙人掌打成浆液,按照100g浆液加入乳酸菌活菌105CFU和酵母菌104CFU,发酵60h,加入75%的乙醇并进行间歇的超声震荡,加入有机溶剂进行萃取,最后旋蒸仪得到仙人掌萃取液。先将仙人掌提取液打成浆液,可以将其中大部分的物质释放,将浆液加入乳酸菌活菌105CFU和酵母菌104CFU,分解其中的纤维素和果胶,将其中的活性物质释放,再通过乙醇将其中的有机物萃取。
对比例5:抗敏修复化妆品,由以下质量百分比组分组成,牛蒡子浓缩提取液4%、玉兰提取液6%、茶多酚2%、仙人掌萃取液4%、玻璃苣籽油2.5%、银耳多糖0.4%、SOD2.5%、肌肽2%、L-苹果酸3.6%、蜂胶3%、余量为水。
对比例6:抗敏修复化妆品,由以下质量百分比组分组成,实施例17制得的间充质干细胞提取物15%、玉兰提取液6%、茶多酚2%、仙人掌萃取液4%、玻璃苣籽油2.5%、银耳多糖0.4%、SOD2.5%、肌肽2%、L-苹果酸3.6%、蜂胶3%、余量为水。
对比例7:实施例17制得的间充质干细胞提取物15%、玉兰提取液6%、茶多酚2%、玻璃苣籽油2.5%、银耳多糖0.4%、SOD2.5%、肌肽2%、L-苹果酸3.6%、蜂胶3%、余量为水。
对比例8:受试者不使用任何护肤产品。
试验方法:筛选250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。
250名女性受试志愿者分为5组每组50例,分别为实施例23组和对比例5-8组,其中对照组5为受试者不使用任何护肤产品,实施例23组和对比例5-8组受试者分别使用本发明实施例23和对比例5-7制得的抗敏修复化妆品,早晚洁面后取适量的抗敏修复化妆品涂抹于面部,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,部分受试对比图如图12所示。
皮肤老化评估:通过观察受试者的5个受试区的皱纹来评价(眉间纹、前额皱纹、鱼尾纹、鼻唇沟、唇周皱纹),评判标准:没有皱纹;轻度(有2-3条皱纹,长度<1.5cm);中度(有2-6条浅皱纹,长度<3cm);重度(有数条主皱纹,长度达4cm,同时伴有浅皱纹)。
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。
在第60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3-4所示。
表3不同化妆品对受试者脸部安全性评估
表4不同化妆品对受试部位的含水量、油脂含量
由表3及表4可知,本发明的抗敏修复化妆品可以改善皮肤暗黄、红斑,提高皮肤角质层含水量及油脂含量。
将实施例23中脐带间充质干细胞中高分子提取物的抗敏修复化妆品应用于河南某人民医院皮肤科接待激素依赖性皮炎50例;其中有45例敏感肌肤的人伴有不同程度的红血丝。在此期间50人停用其他护肤品后,日常使用本发明的产品3个月到6个月,其中46例均恢复正常皮肤,2例红血丝明显减轻,2例未坚持使用中途放弃。说明本发明的抗敏修复化妆品具有很好的抗敏的效果。
以上所述是本发明的优选实施方式,应当指出,上面结合附图和实施例对本发明作了详细的说明,但是,所属技术领域的技术人员能够理解,在不脱离本发明宗旨的前提下,还可以对上述实施例中的各个具体参数进行变更,形成多个具体的实施例,均为本发明的常见变化范围,在此不再一一详述。
Claims (10)
1.一种脐带间充质干细胞中高分子提取物,其特征在于,包括下述步骤:(1)从脐带组织块或脐血中分离得到脐带间充质干细胞;(2)将步骤(1)得到的脐带间充质干细胞定向分化为皮肤干细胞;(3)将步骤(2)得到的皮肤干细胞收集和裂解,并通过孔径为4~20.0nm分子筛将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。
2.根据权利要求1所述的脐带间充质干细胞中高分子提取物,其特征在于,其由下述步骤提取得到:(1)从脐带组织块中分离得到脐带间充质干细胞;(2)将步骤(1)得到的脐带间充质干细胞定向分化为皮肤干细胞;(3)培养皮肤干细胞,并在对数期采用50~100mJ/cm2紫外辐照细胞6~8h;(4)将步骤(3)得到皮肤干细胞正常培养18~36h后,再置于湿度为80~85%、氧气6~8%、二氧化碳5~6%、余量为氮气的低氧环境下培养18~36h后,收集培养液并裂解得到的皮肤干细胞,并通过孔径为10.0nm分子筛得到活性中高分子物质,将活性中高分子物质进行富集得到脐带间充质干细胞中高分子提取物。
3.根据权利要求1所述的脐带间充质干细胞中高分子提取物,其特征在于,步骤(1)中,所述脐带间充质干细胞是从脐带组织块或脐血中经过原代培养步骤、干细胞的传代培养步骤、干细胞的冻存步骤、干细胞的复苏与培养步骤和人源脐带间充质干细胞制剂的制备步骤得到的。
4.根据权利要求3所述的脐带间充质干细胞中高分子提取物,其特征在于:在步骤(2)中,所述的间充质干细胞培养基为无血清培养基:所述无血清培养基是在无血清基础培养基中添加终浓度为4.5wt%~5.5wt%的UltraGRO无血清培养基、45μg/ml~55μg/ml的维生素C、15ng/ml~25ng/ml的干细胞生长因子、1.5mmol/ml~2.5mmol/ml的L-谷氨酰胺、90ng/ml~110ng/ml的人转化生长因子TGF-β1、15ng/ml~25ng/ml的表皮生长因子和15ng/ml~25ng/ml的人纤维细胞生长因子、黄芩甙元0.1~0.3mg/L。
5.根据权利要求2所述的脐带间充质干细胞中高分子提取物,其特征在于:在步骤(4)中,所述皮肤干细胞采用冰浴超声破碎对皮肤干细胞进行裂解,冰浴超声破碎条件为间歇超声,超声破碎的功率300~350W,每工作10s,间歇3s,循环至少20次。
6.一种含有如权利要求1-5任意一项所述的脐带间充质干细胞中高分子提取物的抗敏修复化妆品,其特征在于:包括如下百分含量的原料组成:脐带间充质干细胞中高分子提取物13~18%、牛蒡子浓缩提取液3~5%、玉兰提取液5~8%、茶多酚1~3%、仙人掌萃取液3~5%、玻璃苣籽油1~3%、银耳多糖0.3~0.5%、SOD 2~3%、肌肽1~3%、L-苹果酸3~4%、蜂胶3%、余量为水。
7.根据权利要求6所述的脐带间充质干细胞中高分子提取物的抗敏修复化妆品,其特征在于:所述牛蒡子浓缩提取液的制备方法包括如下步骤:将牛蒡子粉碎过40目筛网,采用并放入采用逆流浸提柱法提取牛蒡子中有效成分,得到牛蒡子提取液,并取出牛蒡子渣加入质量浓度为70~80%的酒精,按1:10~15的固液质量比在75~85℃下提取3次,合并得到提取液;提取液经减压蒸干至恒重,得到牛蒡子浓缩提取物。
8.根据权利要求6所述的脐带间充质干细胞中高分子提取物的抗敏修复化妆品,其特征在于:所述玉兰提取液的制备方法包括如下步骤:取新鲜玉兰叶洗净、沥干,通过粉碎机将玉兰叶打至成糊状,按1g叶浆加3~5mL体积浓度为75%的乙醇并进行间歇的超声震荡,每超声震荡3min,停歇1min,重复4~6次。
9.根据权利要求6所述的脐带间充质干细胞中高分子提取物的抗敏修复化妆品,其特征在于:所述仙人掌萃取液的制备方法如下:仙人掌打成浆液,按照100g浆液加入乳酸菌活菌105CFU和酵母菌104CFU,发酵48~72h,加入75%的乙醇并进行间歇的超声震荡,加入有机溶剂进行萃取,最后旋蒸仪得到仙人掌萃取液。
10.一种如权利要求1-5任意一项所述的脐带间充质干细胞中高分子提取物的检测方法:其特征在于,采用反相液质联用方法。
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