CN108096278A - 一种人间充质干细胞活性成分乌发产品的制备方法 - Google Patents
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Abstract
本发明公开了一种人间充质干细胞活性成分乌发产品的制备方法。该方法使人胎盘来源的间充质干细胞经体外无血清、低氧条件培养后,使其分泌的活性成分FGF的表达量上升,浓度大于等于25mg/ml。经体外的离心、冻融和浓缩富集后,所收获的活性组分可以用于乌发产品的开发和制备。
Description
技术领域
本发明涉及一种人间充质干细胞分泌活性组分的提取和富集方法,利用人胎盘间充质干细胞分泌活性成分制备乌发产品,用于白发的改善。
背景技术
白头发是指全部或部分变白的头发。生白发是由于头发髓质和皮质里黑色素颗粒减少或被空气填空的缘故,其原因有先天性和后天性两种。正常情况下,毛乳头内有丰富的血管,为毛乳头、毛球部提供充足的营养,黑色素颗粒便顺利合成。当黑色素颗粒在毛乳头、毛球部的形成发生障碍,或虽然形成但因某种因素,不能运送到毛发中去,从而使毛发髓质、皮质部分的黑色素颗粒减少、消失时,就会出现白发。随年龄增长,黑色素细胞的数量会减少(每10年减少10~20%)。40岁后这种减少会很明显,尤其体现在头发上。
“人类基因组计划”之一摩根洛科学家首次发现,在头发的根部存在一种负责制造成纤维细胞生长因子(Fibroblast growth factors,FGF)的基因,称之为“FGF基因”,这是影响黑发生长的根源。FGF黑发基因是一种能够促使毛囊中的色素干细胞向发根底部移动,从而使色素干细胞释放出更多的黑色素的基因片段。当该基因数量减少,功能降低,黑素细胞就会减少,同时抑制毛发细胞自杀行为的能力也会减弱。进一步研究后发现,把“FGF”蛋白质添加到黑素细胞上去后,有抑制细胞自杀作用的Bc12基因数量可增加5倍。同时,相关的研究也显示黑色素的形成过程,是由酪氨酸酶氧化酪氨酸而成的,也就是说,黑色素形成的基础是酪氨酸,酪氨酸缺乏也会造成少白头。
间充质干细胞能够分泌毛囊干细胞所需黑发源FGF。随着FGF基因组工程和蛋白组工程(结构、功能和作用机制)的研究深入,间充质干细胞提取的FGF被作为细胞增殖因子、血管形成因子、神经营养因子、形态发生因子、组织修复-再生因子,主要运用于发育学、生理学和临床药理学等方面。
从检索的文献和专利(包括授权和公开未授权)情况来看,乌发产品的研究主要是有两个方面:专利号CN106913866A[P].2017.、CN106109706A[P].2016.、CN104306192A[P].2015.等都是采用有乌发效果的中药材进行产品研究。采用有富含酪氨酸的食疗方式进行乌发研究。
2001年,利根川博士全球研发中运用名贵中药材提取FGF蛋白并结合纳米技术,研究出FGF纳米黑发精华修护素,此黑发产品目前被公认为全球首款根源性黑发产品,这是目前全球利用FGF蛋白研究的唯一乌发产品。
利用人源间充质干细胞分泌的FGF及酪氨酸的乌发产品有可能从根源上解决白发带来的困扰。
发明内容
本发明提供一种独特的人胎盘间充质干细胞活性成分乌发膜的制备方法,即富含人胎盘间充质干细胞分泌FGF和酪氨酸结合的乌发膜。其特征在于,该方法通过体外培养条件参数的调控,使人胎盘间充质干细胞分泌的FGF的表达量上升,并经过有效提取后,单独使用或与富含酪氨酸的成分(如但不限于马铃薯皮浸泡液)配比制成乌发产品。
*除非另有说明,本发明中所采用的百分数均为重量百分数。本发明的目的通过下述技术方案予以实现:
用组织贴壁法或组织研磨法从胎盘或脐带获取间充质干细胞,置37℃、5%CO2静止培养,当细胞汇合度达到80%时,弃上清,用20mM PBS清洗细胞3次,更换为无血清培养基,于37℃、1% O2条件下静止培养40小时后,收获培养上清液,于4℃,1000g离心5分钟去除细胞碎片,0.22μm滤膜过滤,经12000g离心2小时,沉淀用PBS重悬,检测FGF的浓度。以25pg/ml的浓度涂抹于毛囊根部。
本发明提供了一种含有人胎盘间充质干细胞成分的乌发产品,提供了一种条件培养人胎盘间充质干细胞,以及培养上清中有效成分FGF的富集和检测。同时,还提供了与土豆皮浸泡液联合使用,制备乌发液的方法。
其中,所述的人间充质干细胞主要来源于人胎盘附属组织,比如羊膜、脐带。
与现有的技术相比,本发明具有以下有益效果:
本发明所制备的人胎盘间充质干细胞组分含有乌发因子FGF,与目前采用多种天然名贵植物提取的乌发因子FGF或者基因工程技术合成的乌发因子FGF相比,能促使毛囊中的色素干细胞向发根底部移动,从而使色素干细胞释放出更多的黑色素;同时间充质干细胞分泌成分中含有酪氨酸,酪氨酸是黑色素形成的基础,本发明提供的乌发膜乌发效果快,并且能够改善病理性少年白和生理性老年白。
人胎盘间充质干细胞上清液分泌物除了含有乌发因子FGF,还分泌表皮生长所需要的VEGF、PDGF,神经营养因子BDNF、3(NT-3),能减轻各种原因导致的头皮损伤、减轻炎症反应,从而改善毛乳头、毛球部功能。
具体实施方式
下面结合具体实施例,对本发明作进一步清楚地说明,但它们并不是对本发明保护范围的限定。
实施例1
——人胎盘间充质干细胞分泌组分乌发液的制备,采用以下步骤:
主要包括脐带、羊膜来源的间充质干细胞,胎盘来源MSC细胞主要包括脐带、羊膜来源的间充质干细胞,培养采用以下步骤进行:
脐带、羊膜的处理:
脐带包括一条静脉和两条动脉,周围是华通氏胶(Wharton’s Jelly),外层由羊膜来源的上皮包裹,从发育角度来说,脐带是干细胞形成及所经通路,已有研究表明人脐带的结缔组织是间充质干细胞丰富的组织来源。处理方法是首先剥离脐带动静脉和外层上皮,留下凝胶状组织(华通氏胶),切除双侧带夹痕及淤血的部分。
羊膜是胎膜最内层,胎盘包裹胎儿面的一层薄而半透明的膜,由胚胎羊膜囊壁发育而成,在胎盘面与绒毛膜相贴,由人羊膜间充质细胞和人羊膜上皮细胞组成,其表面没有神经、血管、肌肉和淋巴等组织。
消毒 从储液瓶中取出胎盘脐带或羊膜(运输储液:DMEM/F12+1%的双抗50~150ml; 运输储存条件4℃),先用75%酒精对组织进行消毒擦拭,然后用漂洗液(不含钙镁但含有双抗的无菌PBS)反复冲洗组织,直到洗净残余血细胞。
组织块处理 脐带组织处理: 用PBS洗3 次,去掉残留的血细胞。把脐带剪切成2.0 cm左右的片段并剔除血管(1 条脐静脉,2条脐动脉),留下内膜,内膜即为华通氏胶,将其剪成大小约1 mm3的组织块(细碎小块;1mm3大小;呈肉糜状,以能用吸管吸取为标准)。羊膜组织处理:用已干烤灭菌的组织剪及手术剪将羊膜剪成大的组织块,一边将大的组织块剪成小块,一边用手术镊或者虎齿镊夹持进行再次进行漂洗,避免溢出,如此重复3遍,至漂洗液清亮,弃漂洗液,用1ml移液枪或者移液器吸尽剩余的PBS。-此操作在无菌培养皿里进行操作。加入含1%的双抗的DMEM/F12到无菌培养皿,继续用手术剪将组织剪碎(细碎小块;1mm3大小;呈肉糜状,以能用吸管吸取为标准)。
组织块悬液配制及分装 补加含1%的双抗的DMEM/F12终体积27ml,加入终浓度为10%的胎牛血清3ml,用5ml移液枪或者移液器反复吹打组织块悬液,然后按10ml/T25细胞培养瓶进行分装。将T25瓶放入CO2培养箱进行培养,设置培养条件:5%CO2浓度、37℃;根据细胞爬出贴壁情况从3天、5天、7天进行更换组织块培养液(换液时小心吸取原培养液更换相同体积的培养液),每天观察细胞汇合度情况。
MSC传代培养当细胞达80%以上汇合后,每个T25瓶加入2~3ml 0.25% Trypsin-EDTA胰酶消化(加入胰酶后转动T25瓶,让胰酶平铺浸润细胞面10s,立即倾去胰酶,放置30s, 轻轻拍打T25瓶,立即加入10ml组织块培养液终止消化),按1∶2的比例传代到T75瓶(含50ml组织块培养液),继续扩增培养。每天观察细胞生长和汇合度情况。
控制细胞代次低于P10 ,当细胞汇合度达到80%时,弃上清,用20mM PBS清洗细胞3次,更换为无血清培养基(如但不限于OptiMEM),于37℃、1% O2条件下静止培养40小时后,收获培养上清液,于4℃,1000g离心5分钟去除细胞碎片,0.22μm滤膜过滤,于室温,-80℃反复冻融3次,灌装于西林瓶中在真空度下进行冻干(-70℃,24h)后储存。
使用时,参照FGF浓度25pg/ml,用含有1%橄榄油的生理盐水或注射用水溶解后,涂抹于毛囊根部。
实施例2
——人胎盘间充质干细胞外泌体组分乌发液的制备,采用以下步骤:
人胎盘间充质干细胞的培养采用和实施例1相同的步骤至步骤5,
控制细胞代次低于P10 ,当细胞汇合度达到80%时,弃上清,用20mM PBS清洗细胞3次,更换为无血清培养基(如但不限于OptiMEM),于37℃、1% O2条件下静止培养40小时后,收获培养上清液,于4℃,1000g离心5分钟去除细胞碎片,0.22μm滤膜过滤,经12000g离心2小时,沉淀用PBS重悬,使用时,参照FGF浓度25pg/ml,用含有1%橄榄油的生理盐水或注射用水溶解后,涂抹于毛囊根部。
实施例3
——人胎盘间充质干细胞分泌体组分与马铃薯皮浸泡液乌发产品的制备,采用以下步骤:
人胎盘间充质干细胞的培养采用和实施例1相同的步骤至步骤6,
新鲜马铃薯皮洗净、切碎,研磨成泥,用20mM PBS浸泡1小时,离心,上清液用0.45μm的滤膜过滤。
使用时将制备的人胎盘间充质干细胞分泌液组分冻干粉按FGF浓度25pg/ml溶解于马铃薯皮过滤液中,涂抹于毛囊根部。
实施例4
——参考中国生物制品规程以及新发布的《干细胞通用要求》进行间充质干细胞收获液及间充质干细胞破碎液的检定,
生物学特性测定
生物学特性检测包括标志物,分化潜能,细胞形态,遗传学及代谢酶亚型谱的检测。
安全性检测
安全性检测包括无菌试验(细菌、真菌)、支原体,用酶联免疫试剂盒对细胞内外源致病因子(包括艾滋病、梅毒、传染性肝炎)、内毒素等进行检测结果均为阴性。用酶联免疫法检测,牛血清白蛋白残留量应不高于50ng/mL。检测结果的平均值为20ng/mL。
稳定性
胎盘组织优选无传染病、慢性病、遗传病,同时无吸烟、酗酒、孕期年龄适中的父母,具有完备的伦理手续,相关的母亲临床检测结果数据存档备案,与培养后的间充质干细胞检测进行比对。分离的间充质干细胞需要进行密度、浓度、纯度、存活率和生物学活性的检测。
有效性
用去垢剂相容性蛋白浓度检测法(Bio-Rad)、或ELISA等方法人胎盘对间充质干细胞分泌的相关细胞因子浓度进行测定, 主要测定指标为FGF、其它细胞因子包括但不限于VEGF、PDGF、BDNF、3(NT-3)。结果显示,FGF的表达量为25pg/ml。
实施例5
——人胎盘间充质干细胞组分乌发效果的动物模型的建模,采用以下步骤:
如上实施例所制备的间充质干细胞分泌组分、马铃薯皮浸泡液、生理盐水、10%水合氯醛溶液、30%过氧化氢(脱色素)
实验动物:健康纯黑天竺豚鼠40只(300-350克),雌雄兼用
取健康纯黑天竺豚鼠40只,用剪刀剪去豚鼠背部黑色毛区长毛,再用剃毛器剃去短毛,不可损伤皮肤,面积5cmX5cm.按体重随机分成4组,每组10只,分别为A组:正常对照组;B组:模型对照组;C组:人胎盘间充质干细胞分泌组分;D组:对应的溶解间充质干细胞分泌组分的溶剂。B-D组均用5%过氧化氢外涂豚鼠受试区40天(毛发变为白色),每天2次,每次用2ml。在此期间,每天剔毛一次。造模实验结束后,每组随机取1只豚鼠切取适当大小的实验区皮肤组织作病理切片,观察皮肤组织和毛囊的黑色素变化。
以上4组豚鼠,造模成功后,A组不做处理;B组外涂生理盐水;C组间充质干细胞分泌的活性组分。每日涂抹2次,每次2ml,连续涂抹40天。在此期间,每3天剔毛一次
实验结束后,肉眼观察各实验组的改善效果。选取用药部位中心4cm2处为一观察单位判定效果,其标准如下:“优”为观察区色素恢复面积和新生毛中黑毛≧75%;“良”为观察区色素恢复面积和新生毛中黑毛≧50%;“中”为观察区色素恢复面积和新生毛中黑毛≧25%;“差”为观察区皮肤苍白或呈白斑状,无毛生长或者新生白毛。总有效率以“优”加“良”计。
最后切取适当大小的实验区皮肤组织,用10%甲醛固定,进行常规组织脱水,石蜡包埋,HE染色,光镜镜检,观察豚鼠皮肤组织与毛囊黑色素生长相关的指标:一般组织学检查。真皮厚度测量:毛发生长期皮肤增厚,故测定真皮厚皮(基底膜至基膜间的距离)可间接反应毛发生长状况。每例侧3次真皮厚度(40倍),取其平均值,并进行统计学处理。毛囊计数:每例数3个高倍视野(200倍)的毛囊数,取其平均值,并进行统计学处理。真皮层毛细血管数:每例数3个高倍视野(200倍)的毛细血管数,取其平均值,并进行统计学处理。在高倍镜下(400倍)计数每100个基底细胞中含黑色素粒的细胞数,并进行统计学处理。在高倍镜下(400倍)随意观察50个毛囊,计算其中有黑色素毛囊数,并进行统计学处理。
统计学处理:实验数据以均值±标准差(X±S)表示,采用统计学软件包SPSS17.0进行统计学分析。单项有序分类资料的比较用Ridit分析,数值资料的相互比较用方差分析,P〈0.05表示差异具有显著性意义。
一般组织学检查:检查结果被覆鳞状上皮完整,角化层无增厚,真皮内无血管扩张充血、炎细胞浸润等,表示豚鼠皮肤结构完整。
间充质干细胞分泌活性成分改善毛发早白的效果观察发现,含黑色素粒的细胞数与对照组相比,数量增加30%,具有显著的统计学意义(P<0.05),对豚鼠皮肤组织学没有影响。
实施例6
——人胎盘间充质干细胞分泌组分提取液和中药乌发产品的配合使用,采用以下步骤:
人胎盘间充质干细胞分泌组分提取液按实施例1和实施例2制备
使用时,人胎盘间充质干细胞分泌组分提取液按1%-50%的比例添加到中药乌发产品联合使用。
Claims (4)
1.一种人间充质干细胞活性成分乌发产品的制备方法,其特征在于,用低氧、无血清的条件对体外培养的人间充质干细胞进行调控,使分泌的成纤维生长因子FGF的表达量上升,并通过体外离心、冻融、浓缩、过滤等方法提取到富含FGF的组分。
2.提取到的分泌活性组分可以单独或者组合其它乌发成分使用。
3.根据权利要求1的制备方法,其特征在于,使用无血清和低氧条件作为调控参数,使分泌的FGF等细胞因子的浓度增高。
4.根据权利要求1和权利要求2的方法,所制备的分泌活性组分中FGF的浓度大于等于25pg/ml。
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