CN116211891A - 一种人间充质干细胞条件培养液冻干粉制剂及其制备方法 - Google Patents
一种人间充质干细胞条件培养液冻干粉制剂及其制备方法 Download PDFInfo
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Abstract
本发明涉及医药美容领域，具体涉及一种人脐带间充质干细胞条件培养液冻干粉制剂及其制备方法。该美容制剂包括以下重量份的原料组成：人脐带间充质干细胞条件培养液90%～96%，甘露醇5%～8%，寡肽0.4%～0.6%，所述的人脐带间充质干细胞条件培养液是从人脐带来源的间充质干细胞在培养过程中收集的上清液，由辽宁医学诊疗科技研发中心有限公司提供；培养液为人间充质干细胞无血清培养基，培养基的pH为7.0±0.5，渗透压为280‑340mOsmol/kg。本发明在抑制皮肤老化、美白肌肤、修复皮肤损伤等诸多方面均有较显著的效果，可实现对肌肤状态的精准调节，从而使肌肤细胞年轻化。
Description
技术领域
本发明涉及医药美容领域，具体涉及一种人间充质干细胞条件培养液冻干粉制剂及其制备方法。
背景技术
间充质干细胞（Mesenchymal stem cells ,MSCs）是一类具有自我复制能力和多向分化潜能的非造血祖细胞，广泛存在于骨髓、脂肪、肝脏、脐带、脐带血、胎盘等组织中。
干细胞具有多向分化和自我更新能力。多种干细胞，如造血干细胞、胚胎干细胞（ESC）、间充质干细胞（MSC）、诱导多能干细胞、脂肪干细胞，已被用于临床细胞治疗或临床研究，而MSC相对于其他种类干细胞具有明显的优势，因其来源丰富、易于获得、低免疫原性且不存在伦理限制。研究表明，MSC治疗能降低宿主组织细胞内的氧化应激水平。
MSC分泌多种细胞因子，其中白细胞介素6（IL-6）、IL-8、脑源性神经营养因子（BDNF）、肝细胞生长因子（HGF）表达量较多，还包括TNF-α、血管内皮生长因子（VEGF）、白血病抑制因子（LIF）、胰岛素样生长因子1（IGF-1 ）、表皮生长因子、成纤维细胞生长因子（FGF）、转化生长因子β（TGF-β）等因子。
外泌体是细胞在不同应激状态下分泌的直径为40～100 nm的囊泡状小体，起源于细胞的核内体系统，首先细胞经胞吞作用内陷形成早期核内体，随后核内体膜再次内陷形成腔内小囊泡，并选择性地接受胞浆内的蛋白质与脂质而形成晚期核内体，随后晚期核内体膜与细胞膜融合后以胞吐作用将大量小囊泡释放到细胞外，即为外泌体。MSC分泌外泌体的过程与其它细胞相同，也是通过这样一个“内陷－融合－外排”的过程分泌的。2010年Lai等首次分离间充质干细胞来源的外泌体（Mesenchymal stem cell derived exosomesMSC-Exo）。后续研究表明，其具有重要的生物学功能。
多项干细胞移植的研究表明，干细胞在受植床的移植率和成活率均较低，干细胞作用的机制更多倾向于其释放可溶性分子的旁分泌效应参与伤口愈合过程。因此，间充质干细胞生长过程中分泌到培养液中的生长因子、细胞因子的功能越来越受到重视。近年来，通过收集间充质干细胞生长过程中的培养液，经过过滤、浓缩等处理，去除细胞成分，获得含有更高浓度生长因子和细胞因子的活性液体，称为条件培养液，用于基础研究和某些疾病的治疗获得了较好的效果。间充质干细胞条件培养液中不含有细胞，相比于间充质干细胞移植，应用间充质干细胞条件培养液要安全得多。最近的临床研究报道应用骨髓间充质干细胞条件培养液没有出现任何系统性和局部并发症。
皮肤位于人体表面,由表皮、真皮和皮下脂肪组织组成,是人体最大器官。它不仅具有十分重要的屏障作用和防御功能,而且还具有诸多的生理作用和代谢功能。ROS是在细胞代谢、氧气消耗的关键过程中产生的一类具有高活性的小分子化合物，包括超氧阴离子（O2-）、过氧化氢（H2O2）和羟基自由基（HO-）等。细胞内大部分ROS是由线粒体呼吸链产生；存在金属离子时，也通过芬顿反应（一种非酶促反应）产生部分ROS。皮肤的老化往往伴随着氧化应激水平升高。皮肤老化可以表现为皮肤变薄,皮下脂肪减少,产生皱纹、松弛、粗糙、干燥等。皮肤老化是多种原因共同作用的结果,尽管现阶段对其产生机制知之甚少,但尚有一部分机制已经被证实, 如DNA损伤修复、线粒体机制、细胞凋亡、细胞自噬及氧化损伤等均在皮肤老化过程中起到重要作用。虽然皮肤衰老不能停止,但是减轻这些因素的毒性作用可以减慢衰老。
而MSC培养上清中含有大量的干细胞分泌的生长因子和外泌体，对皮肤有很强的修复、抗衰老作用。利用组织损伤的实验动物模型，移植MSC细胞或注射MSC培养上清，都可以显著改善受损器官的功能，说明MSC部分治疗作用是通过其分泌的可溶性细胞因子实现的；除分泌因子外还包括细胞外囊泡、外泌体等。
衰老和疾病发生发展大多伴随氧化应激水平升高干细胞治疗已应用于多种疾病或其临床研究，而间充质干细胞（MSC）相对于胚胎干细胞和诱导多能干细胞具有明显的优势。
冻干粉(freeze-dried powder)为固体干粉剂, 能在常温下长期保存,性能稳定,输注方便,保存费用低廉,液体状态下的间充质干细胞条件培养液在室温保存时间短，不利于运输。对其进行冻干操作，制成冻干粉可以保留细胞因子的活性，便于储存于运输。将冻干粉制剂复溶后可以注射到皮肤真皮层，其中的细胞因子可以发挥抗衰老作用。
发明内容
本发明提供了一种人间充质干细胞条件培养液冻干粉及其制备方法，该产品在抑制皮肤老化、美白肌肤、修复皮肤损伤等方面具有显著作用。
本发明的技术方案是：一种人间充质干细胞来源的外泌体美容制剂，主要原料组分设置及其质量分数为：人脐带间充质干细胞条件培养液90%～96%，甘露醇5%～8%，寡肽0.4%～0.6%。
进一步地，所述的人脐带间充质干细胞条件培养液是从人脐带来源的间充质干细胞在培养过程中收集的上清液，由辽宁医学诊疗科技研发中心有限公司提供；培养液为人间充质干细胞无血清培养基,培养基的pH为7.0±0.5,渗透压为280-340mOsmol/kg。
进一步地，所述冻干过程为：
冻干粉针制备：取人脐带间充质干细胞条件培养液（由辽宁医学诊疗科技研发中心有限公司提供），用纱布粗滤，0.22微孔滤膜过滤，加入5%～8%甘露醇冰浴下搅拌至其完全溶解，分装至5ml西林瓶后（2ml/瓶）放置4℃冰箱备用。选用VirTis冷冻真空干燥机（型号wizard 2.0）冻干，机器先进行预冷至5℃后，放入待冻干溶液。将板层温度在 0.5 小时内，匀速降温至-40℃，然后保持4h进行预冻。后续步骤如下：
step1：-35℃ 240min 真空度20
Step2：-30℃ 60min 真空度20
Step3：-25℃ 60min 真空度20
Step4：-15℃ 60min 真空度20
Step5：0℃ 60min 真空度20
Step6：10℃ 30min 真空度20
Step7：25℃ 30min 真空度20
Post8：35℃ 240min 真空度20。
附图说明
图1人脐带间充质干细胞条件培养液冻干粉制剂外观形态。
图2人脐带间充质干细胞条件培养液冻干粉制剂扫描电镜图。
图3人脐带间充质干细胞条件培养液冻干粉制剂冻干曲线。
具体实施方式
本发明提供了一种人脐带间充质干细胞条件培养液冻干粉及其制备方法，为使本发明的目的、技术方案和效果更加清楚，以下结合具体实施例对本发明进行进一步的详细说明。应当理解的是，此处所描述的具体实施例仅仅为了解释本发明，并不限定于本发明。实施例中所用到的各种常用试剂，均为市售产品。
实施例1
取人脐带间充质干细胞条件培养液100g，用纱布粗滤，0.22微孔滤膜过滤，加入5g的甘露醇、0.4g的寡肽。冰浴下搅拌至其完全溶解，分装至5ml西林瓶后（2ml/瓶）放置4℃冰箱备用。选用VirTis冷冻真空干燥机（型号wizard 2.0）冻干，机器先进行预冷至5℃后，放入待冻干溶液。将板层温度在 0.5 小时内，匀速降温至-40℃，然后保持4h进行预冻。后续步骤如下：
step1：-35℃ 240min 真空度20
Step2：-30℃ 60min 真空度20
Step3：-25℃ 60min 真空度20
Step4：-15℃ 60min 真空度20
Step5：0℃ 60min 真空度20
Step6：10℃ 30min 真空度20
Step7：25℃ 30min 真空度20
Post8：35℃ 240min 真空度20。
实施例2
本实施例提供了一种脐带间充质干细胞因子冻干粉制剂的制备方法，与实施例1相比，加入1g甘露醇。其余步骤与实施例1相同。
实施例3
本实施例提供了一种脐带间充质干细胞因子冻干粉制剂的制备方法，与实施例1相比，加入2g甘露醇。其余步骤与实施例1相同。
实施例4
本实施例提供了一种脐带间充质干细胞因子冻干粉制剂的制备方法，与实施例1相比，加入3g甘露醇。其余步骤与实施例1相同。
实施例5
不同浓度甘露醇效果评价对实施例1～4中的脐带间充质干细胞因子冻干粉的外观、色泽、复水溶性按表1标准进行评价。
表1脐带间充质干细胞因子冻干粉的外观、色泽、复水溶性标准
表2不同甘露醇含量对外观与色泽，复水性、综合评价的结果
如上表所示，冻干保护剂甘露醇浓度为1%、2％、3％时冻干产品形态不佳，出现分层现象，样品萎缩，不饱满，复水溶时间均在120s以上，样品复水溶后呈无色、澄清、透明状，评分低。甘露醇浓度为5％时，冻干产品形态良好，冻干粉末饱满、无分层现象，样品复水溶时间较快，约为10s，样品复水溶后呈无色、澄清、透明状，评分高。
实施例6
人脐带间充质干细胞条件培养液冻干粉制剂的皮肤刺激性实验
试验材料：实施例1中所述的冻干粉制剂
试验对象：健康的三月龄小白鼠
将本发明实施例1制得的冻干粉制剂按照《化妆品安全技术规范》(2015年版)“皮肤刺激性实验方法”进行皮肤刺激性的测定，具体操作和结果如下：
取6只小鼠，将实施例1的产品按照《化妆品卫生规范》要求涂在背部脱毛皮肤上(去毛范围为3cm×3cm，涂抹面积为2.5cm×2.5cm)，连续涂抹10h，以另外6只小鼠相同方法脱毛后不涂抹该产品皮肤作为对照，实验结束后用清水清洗，于清洗后的1、24、48和72h观察涂抹部位皮肤反应，6只小鼠与未涂抹组一样均未出现红斑、水肿，证明本发明产品没有皮肤刺激性。
实施例7
人脐带间充质干细胞条件培养液冻干粉制剂的皮肤测试
试验材料：实施例1中所述的冻干粉制剂
试验对象：健康无皮肤过敏史的50名年龄为20-45岁的女性。
实施步骤：取实施例1中的冻干粉制剂复苏液，涂抹在面部皮肤，每天早晚涂抹相应样品2次，在连续使用30d后，观察受试者皮肤状况。
结果显示，受试者使用实施例1中的人脐带间充质干细胞条件培养液冻干粉后，皮肤粗糙度显著下降，皮肤弹性增强，肤色更均匀，毛孔减小，面部状况有明显改善。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
Claims (3)
1.一种人脐带间充质干细胞条件培养液冻干粉制剂及其制备方法，其特征在于：原料组分设置及其质量分数为：人脐带间充质干细胞条件培养液90%～96%，甘露糖醇5%～8%，寡肽0.4%～0.6%。
2.根据权利要求项1所述的一种人脐带间充质干细胞条件培养液冻干粉制剂及其制备方法，其特征在于：所述的人脐带间充质干细胞条件培养液是从人脐带来源的间充质干细胞在培养过程中收集的上清液，培养基为人间充质干细胞无血清培养基,培养基的pH为7.0±0.5,渗透压为280-340mOsmol/kg。
3.根据权利要求项1所述的一种人间充质干细胞来源的外泌体美容制剂，其特征在于：冻干过程为：取人脐带间充质干细胞条件培养液，用纱布粗滤，0.22微孔滤膜过滤，加入5%～8%甘露醇冰浴下搅拌至其完全溶解，分装至5ml西林瓶后（2ml/瓶）放置4℃冰箱备用；选用VirTis冷冻真空干燥机（型号wizard 2.0）冻干，机器先进行预冷至5℃后，放入待冻干溶液；将板层温度在 0.5 小时内，匀速降温至-40℃，然后保持4h进行预冻；后续步骤如下：
step1：-35℃ 240min 真空度20
Step2：-30℃ 60min 真空度20
Step3：-25℃ 60min 真空度20
Step4：-15℃ 60min 真空度20
Step5：0℃ 60min 真空度20
Step6：10℃ 30min 真空度20
Step7：25℃ 30min 真空度20
Post8：35℃ 240min 真空度20。
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