CN113144221B - 外泌体制剂及其制备方法和应用 - Google Patents
外泌体制剂及其制备方法和应用 Download PDFInfo
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- CN113144221B CN113144221B CN202110286975.5A CN202110286975A CN113144221B CN 113144221 B CN113144221 B CN 113144221B CN 202110286975 A CN202110286975 A CN 202110286975A CN 113144221 B CN113144221 B CN 113144221B
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Abstract
本发明涉及一种外泌体制剂及其制备方法和应用,制备方法包括以下步骤:培养基因组中外源转入人成纤维细胞生长因子2基因的诱导多能干细胞,并收集细胞培养液;从所述细胞培养液中提取得到外泌体溶液;在所述外泌体溶液中加入3‑羟基丁酸的可溶性盐,得到所述外泌体制剂。本发明制备得到的外泌体制剂可以显著提高皮肤的抗衰能力,在去皱、美白、淡斑、增加弹性等方面都具有很好的效果,而且成分比较天然,不易过敏和引起免疫反应,成分也容易被人体吸收和降解,临床应用非常安全有效。
Description
技术领域
本发明涉及生物医药技术领域,特别是涉及一种外泌体制剂及其制备方法和应用。
背景技术
外泌体(Exosome)是由活细胞分泌的直径约为30nm~150nm的小囊泡,具有典型的脂质双分子层结构,存在于细胞培养上清液、血清、血浆、唾液、尿液、羊水以及其它生物体液中。外泌体内含有与细胞来源相关的蛋白质rRNA和microRNA,外泌体可通过细胞膜受体直接激活受体细胞,也可运输蛋白质、mRNA、miRNA、lncRNA、circRNA,甚至细胞器进入受体细胞,参与细胞间通讯。外泌体在免疫应答、炎症反应、血管生成、凋亡、凝血和废物处理等生理过程发挥关键作用。外泌体不仅在细胞与细胞间的物质和信息传递中起重要作用,更有望成为多种疾病的早期诊断标志物。
干细胞外泌体里面含有丰富的细胞因子,比如VEGF、FGF等,可以参与皮肤组织修复与再生的各个过程,通过促进皮肤细胞的增殖迁移,促进血管新生,调节免疫反应来促进创伤愈合与皮肤组织再生,对皮肤组织的修复和抗衰老等有显著效果。近年来干细胞及相关组分在皮肤抗衰和提拉紧致等方面的应用也是研究的热点。但是目前一般的制备方法制备得到的外泌体制剂,其保持皮肤紧致和抗衰除皱等效果较差,而且容易引起过敏或免疫排斥反应。
发明内容
基于此,有必要提供一种保持皮肤紧致和抗衰除皱效果较好、不易引起过敏和免疫排斥反应的外泌体制剂的制备方法。
一种外泌体制剂的制备方法,包括以下步骤:
培养基因组中外源转入人成纤维细胞生长因子2基因的诱导多能干细胞,并收集细胞培养液;
从所述细胞培养液中提取得到外泌体溶液;
在所述外泌体溶液中加入3-羟基丁酸的可溶性盐,得到所述外泌体制剂。
本发明外的制备方法采用基因组中掺有人成纤维细胞生长因子2(FGF2)基因的诱导多能干细胞进行细胞培养,然后从收集到的细胞培养液中提取外泌体溶液,并进一步添加3-羟基丁酸的可溶性盐如3-羟基丁酸钠,从而制备得到外泌体制剂。该制备方法的有益效果在于:人诱导多能干细胞本身分泌的外泌体总量以及其中的各种生长因子如VEGF、FGF等有益成分的总量相比于成体干细胞和间充质干细胞等更高,而通过诱导多能干细胞过表达人成纤维细胞生长因子2可以分泌得到更高浓度的天然人成纤维细胞生长因子2,因此制备得到的外泌体制剂中外泌体含量更高,人成纤维细胞生长因子2和其他生长因子等有益成分的总量也更高;βFGF(人FGF2)是FGF家族中很重要的一员,具有抗皱、抗衰、美白祛斑、晒后修复改善肤色、消除深层皱纹、紧致肌肤等功能,同时可促进成纤维细胞产生胶原蛋白;加入的3-羟基丁酸的可溶性盐可以协同各生长因子促进脂肪干细胞和成纤维细胞的增殖,促进脂肪干细胞向脂肪组织分化等;诱导多能干细胞分泌表达得到的人成纤维细胞生长因子2等有益成分比较天然,3-羟基丁酸的可溶性盐的降解产物为二氧化碳和水,很容易被人体吸收和降解,也非常安全,不易过敏和引起免疫反应,临床应用非常安全有效。
在其中一个实施例中,所述诱导多能干细胞的培养环境为:36℃~38℃恒温箱内,且气体环境中氧气浓度不高于1%。
在其中一个实施例中,所述3-羟基丁酸的可溶性盐为3-羟基丁酸钠,所述3-羟基丁酸钠的终浓度为5μM~50μM。
在其中一个实施例中,还包括以下步骤:在所述外泌体溶液中加入透明质酸和维生素C。
在其中一个实施例中,所述透明质酸与所述外泌体溶液的体积比为(1~3):1,所述维生素C的终浓度为5ng/mL~50ng/mL。
在其中一个实施例中,从所述细胞培养液中提取外泌体溶液包括以下步骤:去除所述细胞培养液中的细胞碎片,然后转移至截留分子量为10kDa的超滤管中进行离心浓缩,得到所述外泌体溶液。
在其中一个实施例中,所述基因组中掺有人成纤维细胞生长因子2基因的诱导多能干细胞通过以下步骤制备得到:取诱导多能干细胞,转染含有人成纤维细胞生长因子2基因的慢病毒载体,然后进行单细胞培养和筛选,得到所述基因组中掺有人成纤维细胞生长因子2基因的诱导多能干细胞。
在其中一个实施例中,所述含有人成纤维细胞生长因子2基因的慢病毒载体通过PRlenti-puro载体与成纤维细胞生长因子2基因片段连接得到。
在其中一个实施例中,所述转染的方式为电穿孔,电穿孔的条件为520V~560V,脉冲周期为15~25毫秒。
本发明还提供了一种外泌体制剂,其根据如上所述的制备方法制备得到。
本发明还提供了一种护肤品,其含有如上所述的外泌体制剂。
附图说明
图1为实施例1中慢病毒质粒PRlenti-puro-FGF2的构造示意图;
图2为实施例3中低氧环境下的稳定过表达FGF2的诱导多能干细胞株(实验组)与正常氧浓度培养下的稳定过表达FGF2的诱导多能干细胞株(对照组1)以及正常氧浓度培养下的正常的诱导多能干细胞株(对照组2)分泌的上清液中的TGF-β、PGD-F、VEGF和IGF-1的含量的比较;
图3为实施例3中低氧环境下的稳定过表达FGF2的诱导多能干细胞株(实验组)与正常氧浓度培养下的稳定过表达FGF2的诱导多能干细胞株(对照组1)以及正常氧浓度培养下的正常的诱导多能干细胞株(对照组2)分泌的上清液中的FGF2、HGF和EGF的含量的比较;
图4为实施例3中同体积的干细胞上清液分别通过超滤法、超离法、沉淀法和抗体标记法得到的外泌体总量的比较;
图5为实施例4中20μM浓度的3-羟基丁酸钠对人皮肤成纤维细胞的增殖效果测试图;
图6为实施例4中外泌体制剂组与缺外泌体组和缺3-羟基丁酸钠组的制剂面部填充后,在保湿、美白、祛斑、淡化细纹、缩小毛孔和祛痘等指标的评分结果比较。
具体实施方式
为了便于理解本发明,下面将对本发明进行更全面的描述,并给出了本发明的较佳实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
术语解释
“载体(vector)”是指可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时,载体称为表达载体。载体可以通过转化,转导或者转染导入宿主细胞,使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的,包括但不限于:质粒;噬菌粒;柯斯质粒;人工染色体,例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC);噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于,逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。
“诱导性多能干细胞”(Induced pluripotent stem cells,iPSCs)是指通过导入特定的转录因子将终末分化的体细胞重编程的多能性干细胞。分化的细胞在特定条件下被逆转后,恢复到全能性状态,或者形成胚胎干细胞系,或者进一步发育成新个体的过程即为细胞重编程(Cell reprogramming)。分化是基因选择性表达的结果,并没有改变遗传物质,而重编程在某种意义上就是分化的一个逆转。与经典的胚胎干细胞技术和体细胞核移植技术不同,iPSCs技术不使用胚胎细胞或卵细胞,因此没有伦理学问题。
“电穿孔”也叫电转染,是通过高强度的电场作用,瞬时提高细胞膜的通透性,从而吸收周围介质中的外源分子。这种技术可以将核苷酸、DNA与RNA、蛋白、糖类、染料及病毒颗粒等导入原核和真核细胞内。电转染相对其它物理和化学转化方法,是一种有价值和有效的替代方法。电穿孔是功能强大的将核酸、蛋白及其它分子导入多种细胞的高效技术。
本发明一实施例的外泌体制剂的制备方法,其包括以下步骤S1~S3:
S1、培养基因组中外源转入人成纤维细胞生长因子2基因的诱导多能干细胞,并收集细胞培养液;
S2、从上述细胞培养液中提取得到外泌体溶液;
S3、在上述外泌体溶液中加入3-羟基丁酸的可溶性盐,得到外泌体制剂。
本发明外的制备方法采用基因组中掺有人成纤维细胞生长因子2(FGF2)基因的诱导多能干细胞进行细胞培养,然后从收集到的细胞培养液中提取外泌体溶液,并进一步添加3-羟基丁酸的可溶性盐,从而制备得到外泌体制剂。该制备方法的有益效果在于:人诱导多能干细胞本身分泌的外泌体总量以及其中的各种生长因子如VEGF、FGF等有益成分的总量相比于成体干细胞和间充质干细胞等更高,而通过诱导多能干细胞过表达人成纤维细胞生长因子2可以分泌得到更高浓度的天然人成纤维细胞生长因子2,因此制备得到的外泌体制剂中外泌体含量更高,人成纤维细胞生长因子2和其他生长因子等有益成分的总量也更高;βFGF(人FGF2)是FGF家族中很重要的一员,具有抗皱、抗衰、美白祛斑、晒后修复改善肤色、消除深层皱纹、紧致肌肤等功能,同时可促进成纤维细胞产生胶原蛋白;加入的3-羟基丁酸的可溶性盐可以协同各生长因子促进脂肪干细胞和成纤维细胞的增殖,促进脂肪干细胞向脂肪组织分化等;诱导多能干细胞分泌表达得到的人成纤维细胞生长因子2等有益成分比较天然,3-羟基丁酸的可溶性盐的降解产物为二氧化碳和水,很容易被人体吸收和降解,也非常安全,不易过敏和引起免疫反应,临床应用非常安全有效。
在一个具体示例中,诱导多能干细胞的培养环境为:36℃~38℃恒温箱内,维持饱和湿度,且气体环境中氧气浓度不高于1%。例如,可通入体积比分别为94%N2、5%CO2和1%O2的混合气体。如此,通过低氧环境可以促进诱导多能干细胞表达分泌更多的外泌体和各种生长因子。可以理解,人诱导多能干细胞培养和传代用的培养基为无需滋养层的、无酚红的、无血清的、成分明确的人多能干细胞培养基,可以长期维持人诱导多能干细胞的三胚层分化潜能和自我更新能力。
在一个具体示例中,上述3-羟基丁酸的可溶性盐为3-羟基丁酸钠,3-羟基丁酸钠的终浓度为5μM~50μM,优选为15μM~25μM,对相关细胞的增殖和分化具有更加显著的促进作用。
在一个具体示例中,制备方法还包括以下步骤:在上述外泌体溶液中加入透明质酸和维生素C。维生素C可以抑制黑色素形成,达到淡化已经形成的斑点和色素沉着,改善皮肤颜色美白的效果;透明质酸具有超强的保湿和补水功能,可以改善皮肤营养的新陈代谢,使皮肤柔嫩、光滑、增加弹性、防止皮肤老化等。
在一个具体示例中,透明质酸与外泌体溶液的体积比为(1~3):1,优选为(1~1.5):1,维生素C的终浓度为5ng/mL~50ng/mL,优选为15~25ng/mL。
在一个具体示例中,从细胞培养液中提取外泌体溶液包括以下步骤:去除细胞培养液中的细胞碎片,然后转移至截留分子量为10kDa的超滤管中进行离心浓缩,得到外泌体溶液。超滤方法得到的外泌体相比超速离心等其他方法,对外泌体的损耗更小,得到的外泌体含量更高,而且制备相对容易,对仪器的依赖更小,制备成本更低。可选地,上述超滤管为Ultra-15超滤管,但不限于此。
在一个具体示例中,基因组中掺有人成纤维细胞生长因子2基因的诱导多能干细胞通过以下步骤制备得到:取诱导多能干细胞,转染含有人成纤维细胞生长因子2基因的慢病毒载体,然后进行单细胞培养和筛选,得到上述基因组中掺有人成纤维细胞生长因子2基因的诱导多能干细胞。
在一个具体示例中,含有人成纤维细胞生长因子2基因的慢病毒载体通过PRlenti-puro载体与成纤维细胞生长因子2基因片段连接得到,但不限于此。可以理解,载体还可包含基因工程中常用的调控元件或筛选元件,例如嘌呤霉素筛选基因等。
在一个具体示例中,转染的方式为电穿孔,电穿孔的条件为520V~560V,脉冲周期为15~25毫秒。
本发明一实施例的外泌体制剂,其根据如上所述的制备方法制备得到。可以理解,外泌体制剂中还可以根据需要添加其他助剂。本发明制备得到的外泌体制剂可以显著提高皮肤的抗衰能力,在去皱、美白、淡斑、增加弹性等方面都具有很好的效果,而且成分比较天然,不易过敏和引起免疫反应,成分也容易被人体吸收和降解,临床应用非常安全有效。
本发明还提供了如上所述的外泌体制剂在制备用于皮肤美化的产品中的应用。上述皮肤美化可以是抗衰、除皱、美白和去疤痕修复等,但不限于此。
本发明一实施例的护肤品,其含有如上所述的外泌体制剂。可以理解,护肤品中还可以根据需要添加其他助剂。
下面将结合具体实施例对本发明的实施方案进行详细描述。
实施例1构建慢病毒质粒PRlenti-puro-FGF2
PRlenti-puro-GFP质粒来自中国科学院广州生物医药与健康研究所赠送。FGF2序列来自NCBI中人FGF2基因的CDS序列,经碱基全合成得到。PRlenti-puro载体是通过PRlenti-puro-GFP质粒酶切去除GFP获得,PRlenti-puro-FGF2质粒则通过PRlenti-puro载体和FGF2片段连接得到。如图1所示,其为PRlenti-puro-FGF2的构造示意图。
实施例2获得稳定表达人FGF2的诱导多能干细胞株
人iPS细胞为北京大学生命科学院赠送。首先取一个表面贴附处理过的6孔板,然后每孔加入1mL含有2%的基底胶(Matrigel)的DMEM F12培养基在37摄氏度培养箱里包被2小时。取出冻存的细胞数量约为1×106个的人iPS细胞冻存管,在37℃水浴中快速震荡溶解,吸取冻存管里的液体加入15mL离心管,逐滴加入5mL培养基mTeSR1,轻摇混匀,1000rpm离心5min后弃上清,用含10μmol/L Y-27632的mTeSR1重悬混匀,接种于包被好Matrigel的细胞培养皿表面,放入37度培养箱里培养。第2天mTeSR1培养基换液,培养3天左右等细胞密度达到70%~80%进行细胞传代。
细胞传代时吸弃旧培养基。按0.5mL/孔的量,向瓶内加入RESL消化酶,轻轻摇动培养容器,使消化液流遍所有细胞表面,将培养瓶放入37摄氏度培养箱中消化,计时1min。取出培养瓶,吸弃大部分消化酶,留一点消化酶残液,再将培养瓶放入培养箱,计时3min左右。取出6孔板,镜下观察细胞变化,当大部分细胞变亮变圆,且细胞尚未脱离基质或漂起时即可终止消化。用适量分装的含10μmol/L Y-27632的mTeSR1培养基将细胞克隆轻轻从底部吹下来(切勿用力吹打),把细胞悬液移入离心管。吸弃预先包被6孔板的包被液,按传代1:6比例,将细胞悬液均匀分到培养板内,补足培养基到每孔2mL,摇匀。将6孔板放置于二氧化碳恒温恒湿培养箱开始培养。第2天mTeSR1培养基换液,培养3天左右细胞密度达到70%~80%。
取正在培养的人iPS细胞系,37℃Accutase消化酶胰酶消化5min,使用mTeSR1培养基终止消化后,300g离心5min。去上清,计数,并用电解液重悬成1×106个/20μL。向电解液中加入浓度为1μg/μL的PRlenti-puro-FGF2质粒1μL,混匀。使用Celetrix电转仪进行电转,电转条件为540V,20ms 1次脉冲。然后将电转后细胞立即转移到盛有1mL含10μmol/L Y-27632的mTeSR1培养基的预先包被过的12孔板中进行培养。同时旁边做一个对照孔,对照孔里加入未电转的同样数量的单细胞悬液,加入同样体积的培养基进行培养。
转染48h后,电转组和对照组的细胞换液,每隔3天左右换一次液,一直等到电转组和对照组内的细胞形成克隆并逐渐长大。然后换液,并向电转组和对照组的孔板中加入2ug/mL嘌呤霉素,此后1~2天换一次液,并且一直加嘌呤霉素进行筛选。一直等到对照组里的细胞全部凋亡,而电转组内的未被嘌呤霉素杀伤的细胞逐渐长大,并细胞密度达到70%~80%后开始传代。此时的细胞为稳定表达FGF2的诱导多能干细胞株。
实施例3获取外泌体溶液
当稳定表达FGF2的诱导多能干细胞株密度长到70%~80%时,按照实施例2的方法进行正常消化和传代,传代时细胞培养所用的培养基更换为无需滋养层的、无酚红的、无血清的、成分明确的人多能干细胞培养基。传代后的细胞培养板或培养瓶放到37℃恒温箱内,持续通入含94%N2、5%CO2和1%O2的混合气体,维持低氧环境和饱和湿度。每隔24h收集低氧环境下的细胞上清液,并且进行细胞换液重新放到低氧环境下培养。将收集到的上清液混合放到4度冰箱以待进行外泌体提取。
图2和图3为低氧环境下的稳定过表达FGF2的诱导多能干细胞株(实验组)分泌的上清液中的细胞因子含量与正常氧浓度培养下的稳定过表达FGF2的诱导多能干细胞株(对照组1)分泌的上清液中的细胞因子含量以及正常氧浓度培养下的正常的诱导多能干细胞株(对照组2)分泌的上清液中的细胞因子含量的比较。可以看到实验组的上清液中TGF-β、PDG-F、VEGF、FGF2、HGF、EGF等细胞因子的含量均显著高于对照组1和对照组2。
收集30mL左右干细胞上清液,然后使用Ultra-15超滤管(10kDa MWCO)进行外泌体制备。首先使用0.22μm的Millipore无菌滤器对干细胞上清液进行澄清处理,去除细胞碎片等。用DPBS平衡超滤管,4000g离心10min。从超滤管内管及收集管吸走DPBS,向2个超滤管中分别加入15mL样品,4000g离心30min浓缩。倒空收集管,加入14mL生理盐水进行缓冲液置换,温柔吸打样品数次,4000g离心30min。从浓缩管吸回处理好的样品,此时两个超滤管里的浓缩液合并后体积为1mL。
图3为同体积的干细胞上清液超滤方法得到的外泌体总量与其他方法得到的外泌体总量的比较。可以看到超滤法相比超离法、沉淀法和抗体标记法等分离的外泌体含量更高。
实施例4制备外泌体制剂以及志愿者面部填充测试
向实施例3中的1mL外泌体溶液中加入透明质酸(润月雅,华熙生物)1mL,终浓度为20μM的3-羟基丁酸钠(Sigma-Aldrich),终浓度为20ng/mL的维生素C注射液(国药准字H20044375,郑州卓峰制药有限公司),生理盐水补足总体积为5mL。将外泌体制剂装入5mL无菌水光针注射器中密封。同时制备两个对照组,分别是含有同样体积或浓度的透明质酸、3-羟基丁酸钠、维生素C、生理盐水5mL制剂组(即缺少外泌体组)和含有同样体积或浓度的外泌体、透明质酸、维生素C、生理盐水5mL制剂组(即缺少3-羟基丁酸钠组)。
图4为20μM浓度的3-羟基丁酸钠对人皮肤成纤维细胞增殖效果,可以看到,20μM浓度的3-羟基丁酸钠对人皮肤成纤维细胞的增殖具有显著的促进作用。
寻找20个30~50岁的女性志愿者分成两组,每组10个志愿者。第一组志愿者左边面部的眼角、额头、脸颊等真皮层进行填充注射外泌体制剂,右边面部同样部位注射缺少外泌体组制剂。第二组志愿者左边面部眼角、额头、脸颊等真皮层部位注射外泌体制剂,右边面部同样部位注射缺少3-羟基丁酸钠组制剂。14天后回访各志愿者,对各制剂组的保湿、美白、祛斑、淡化细纹、缩小毛孔和祛痘等指标进行评分,各项总分为10分。
图5为外泌体制剂与其他组的制剂面部填充后在保湿、美白、祛斑、淡化细纹、缩小毛孔和祛痘等指标的评分结果比较。可以看到外泌体制剂组在保湿、美白、祛斑、淡化细纹、缩小毛孔和祛痘等指标的评分上面都优于另外两个对照组,说明该外泌体制剂在医疗美容方面具有非常好的应用前景。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (8)
1.一种外泌体制剂的制备方法,其特征在于,包括以下步骤:
培养基因组中外源转入人成纤维细胞生长因子2基因的诱导多能干细胞,并收集细胞培养液;所述诱导多能干细胞的培养环境为:36℃~38℃恒温箱内,且气体环境中氧气浓度不高于1%;
从所述细胞培养液中提取得到外泌体溶液;从所述细胞培养液中提取外泌体溶液包括以下步骤:去除所述细胞培养液中的细胞碎片,然后转移至截留分子量为10kDa的超滤管中进行离心浓缩,得到所述外泌体溶液;
在所述外泌体溶液中加入3-羟基丁酸的可溶性盐,得到所述外泌体制剂。
2.根据权利要求1所述的制备方法,其特征在于,所述3-羟基丁酸的可溶性盐为3-羟基丁酸钠,所述3-羟基丁酸钠的终浓度为5μM~50μM。
3.根据权利要求1所述的制备方法,其特征在于,还包括以下步骤:在所述外泌体溶液中加入透明质酸和维生素C。
4.根据权利要求3所述的制备方法,其特征在于,所述透明质酸与所述外泌体溶液的体积比为1~3:1,所述维生素C的终浓度为5ng/mL~50ng/mL。
5.根据权利要求1~4任一项所述的制备方法,其特征在于,所述基因组中外源转入人成纤维细胞生长因子2基因的诱导多能干细胞通过以下步骤制备得到:取诱导多能干细胞,转染含有人成纤维细胞生长因子2基因的慢病毒载体,然后进行单细胞培养和筛选,得到所述基因组中外源转入人成纤维细胞生长因子2基因的诱导多能干细胞。
6.根据权利要求5所述的制备方法,其特征在于,所述含有人成纤维细胞生长因子2基因的慢病毒载体通过PRlenti-puro载体与成纤维细胞生长因子2基因片段连接得到。
7.一种外泌体制剂,其特征在于,根据权利要求1~6任一项所述的制备方法制备得到。
8.一种护肤品,其特征在于,含有权利要求7所述的外泌体制剂。
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