CN116515734A - 一种促进毛发再生的诱导乳腺上皮细胞外泌体 - Google Patents
一种促进毛发再生的诱导乳腺上皮细胞外泌体 Download PDFInfo
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Abstract
本发明提供了一种促进毛发再生的诱导乳腺上皮细胞外泌体,其获得包括诱导乳腺上皮细胞的获得以及诱导乳腺上皮细胞外泌体的提取两个步骤。本发明中使用的外泌体为成纤维细胞重编程为乳腺上皮细胞的上清液分离提取得到,不含有害化学成分、重金属等;所述外泌体中的生物活性成分既能有效改善头皮组织新陈代谢,也能促进毛囊细胞的再生,从而有效解决脱发以及秃头的问题。
Description
技术领域
本发明属于生物技术领域,尤其是涉及一种促进毛发再生的诱导乳腺上皮细胞外泌体。
背景技术
脱发是指头发从毛囊脱落的现象。正常脱落的头发都是处于退行期及休止期的毛发,但当头发异常或者过多脱落就值得关注。脱发可能受到多种原因影响,目前世界脱发率高达32.14%,脱发人群的数量仍不断增加。脱发虽然不会对患者机体健康有影响,但患者往往承担巨大的精神压力,对患者生活质量产生重大影响。因此治疗脱发问题,现在拥有旷阔的市场前景。
目前脱发的治疗方法,主要采用药物治疗与手术治疗。药物治疗常采取米诺地尔和非那雄胺进行治疗,米诺地尔也是目前治疗脱发的“金标准”,但这两款药物有着不少不良反应,米诺地尔对于脱发有着心绞痛、胸痛(心包炎)、头痛(血管扩张所致)、过敏反应、皮疹、瘙痒。而手术治疗采用毛囊移植的方法,但其高昂的手术费用与较低的成功率成为阻碍其推广的障碍。治疗脱发的关键,是将处于休止期或退化的毛囊细胞,重新产生毛囊活力。
发明内容
有鉴于此,本发明旨在提出一种促进毛发再生的诱导乳腺上皮细胞外泌体,以克服现有技术的不足,将成纤维细胞或表皮细胞重编程获得诱导乳腺上皮细胞的过程中衍生的外泌体可以激活毛囊细胞并因此诱导毛发再生,副作用小,效果明显。
为达到上述目的,本发明的技术方案是这样实现的:
一种促进毛发再生的诱导乳腺上皮细胞外泌体,其获得包括诱导乳腺上皮细胞的获得以及诱导乳腺上皮细胞外泌体的提取两个步骤,其中诱导乳腺上皮细胞通过如下方法获得,将成纤维细胞或表皮细胞以1×104~5×106个细胞的密度接种至60mm的培养皿中,高糖DMEM+体积百分比10%FBS培养8-24h后,更换诱导培养基R,然后置于37℃,5%CO2培养箱继续培养8天,其中每间隔两天更换一次培养基;更换培养基时,将上清液进行收集,用于外泌体的提取。
优选的,所述诱导培养基R,包括Knockout DMEM/F12、N2(100×)、Neurobasal、B27(50×)、Glutamine(100×)以及Repsox(R),其中Knockout DMEM/F12、N2(100×)、Neurobasal、B27(50×)、Glutamine(100×)的体积比为99:1:97:2:1,Repsox(R)的浓度为5-100μM。
优选的,细胞源于人、小鼠、大鼠、兔、猪、羊、山羊、牛或水牛耳部成纤维细胞或表皮细胞。
优选的,成纤维细胞通过如下方法获得,将贴壁状态良好的山羊耳边缘组织块加入到DMEM培养液进行贴壁培养,每2d~3d更换一次培养基待;原代培养的单层细胞在培养皿中汇合度达80-90%即可进行传代,弃去旧培养液,质量百分比0.1%~0.3%胰蛋白酶进行消化,然后高糖DMEM+10%FBS(体积百分比)培养基进行中和。收集细胞悬液,离心1200r/min,1~3min后弃上清,重悬细胞后均匀接种。
优选的,采用试剂盒提取法对诱导乳腺上皮细胞外泌体进行提取。
优选的,对诱导乳腺上皮细胞外泌体进行提取使用的试剂盒为#41201ES50。
优选的,对诱导乳腺上皮细胞外泌体进行提取包括如下步骤,将诱导乳腺上皮细胞的获得阶段收集的上清液,3000~5000×g,离心10~15min,收集上清;每10mL细胞培养上清加入2~3mL外泌体抽提试剂,涡旋振荡混匀1~2min,再置于2-8℃静止2~3h;将装有混合液的离心管于4℃,10000×g离心30min~60min,弃上清,收集沉淀;取30~100μL PBS均匀吹打沉淀物,转移至1.5mL离心管中;将含有外泌体的离心管于2-8℃,12000~15000×g离心2~3min,弃沉淀保留上清液,置于-80℃冰箱保存。
本发明同时提供如上所述的诱导乳腺上皮细胞外泌体在制备促进毛发再生药物或护理品中的应用。
相对于现有技术,本发明所述的促进毛发再生的诱导乳腺上皮细胞外泌体,具有以下优势:
外泌体是由细胞向胞外分泌的囊泡类小体,直径为30~150纳米,具有典型的脂质双分子层膜结构,其将功能性mRNA,微RNA,细胞因子和转录因子传递给靶细胞,并且能够触发再生。
本发明中使用的外泌体为成纤维细胞重编程为乳腺上皮细胞的上清液分离提取得到,作为有效的细胞间转运蛋白,外泌体可促进头发再生;且不含有害化学成分、重金属等;所述外泌体中的生物活性成分既能有效改善头皮组织新陈代谢,也能促进毛囊细胞的再生,从而有效解决脱发以及秃头的问题。
附图说明
图1为诱导乳腺上皮细胞外泌体的粒径分析图;结果显示,外泌体的粒径范围主要在40-100nm之间;
图2为诱导乳腺上皮细胞外泌体的透射电镜图;结果显示外泌体呈现球形,双层膜结构完整,直径主要在30-100nm范围内。比例尺200nm;
图3为诱导乳腺上皮细胞外泌体促进毛发再生过程图。结果显示,外泌体处理后,小鼠背部毛发随着时间延长逐渐增加;
图4为小鼠皮肤HE和Masson染色图。结果显示与未处理组小鼠相比,处理组小鼠毛囊数目显著增加。A.注射外泌体组HE切片;B.注射外泌体组Masson切片;C.注射PBS组(对照组)HE切片;D.注射PBS组(对照组)Masson切片。箭头标注地方为毛囊;比例尺100μm。
具体实施方式
除有定义外,以下实施例中所用的技术术语具有与本发明所属领域技术人员普遍理解的相同含义。以下实施例中所用的试验试剂,如无特殊说明,均为常规生化试剂;所述实验方法,如无特殊说明,均为常规方法。
下面结合实施例来详细说明本发明。
诱导培养基(R)成分:
200mL体系:
一、诱导乳腺上皮细胞外泌体的提取及鉴定
1.1诱导乳腺上皮细胞的获得
将山羊(选择日龄30-60天的)的耳缘边缘组织块,放入含有双抗的PBS缓冲液中冲洗2-3遍,高糖DMEM+10%FBS(体积百分比)培养基中保存。在实验室中进行组织块处理,首先进行酒精消毒,PBS缓冲液中去除毛发和软骨,去除干净后PBS缓冲液清洗三次。将处理后的组织块置于1.5mL离心管中,用眼科剪剪切至合适大小,并均匀铺至60mm的细胞培养皿中,倒置放于培养箱。待组织块贴壁状态良好时,加入DMEM培养液进行贴壁培养,每2d更换一次培养基。待原代培养的单层细胞在培养皿中汇合度达80-90%即可进行传代,弃去旧培养液,0.25%胰蛋白酶(质量百分比)进行消化,然后高糖DMEM+10%FBS(体积百分比)培养基进行中和。收集细胞悬液,离心(1200r/min,3min)后弃上清,细胞重悬后均匀接种。
将成纤维细胞以5×105的密度接种至60mm的培养皿中,高糖DMEM+10%FBS(体积百分比)培养8-24h后,更换诱导培养基R,然后置于37℃,5%CO2培养箱继续培养8天,其中每间隔两天更换一次培养基。更换培养基时,将上清液进行收集,用于外泌体的提取。
1.2诱导乳腺上皮细胞外泌体的提取
采用试剂盒提取法分离提取外泌体。具体方法如下:3000×g,离心10min,收集上清;每10mL细胞培养上清液加入2.5mL外泌体抽提试剂(细胞培养上清外泌体快速抽提试剂盒#41201ES50),涡旋振荡混匀1min,再置于2-8℃静止2h;将装有混合液的离心管于4℃,10000×g离心60min,弃上清,收集沉淀;取100μL PBS均匀吹打沉淀物,转移至1.5mL离心管中;将含有外泌体的离心管于4℃,12000×g离心2min,弃沉淀保留上清液,置于-80℃冰箱保存。
1.3外泌体的鉴定
通过Zetasize粒度仪,用动态光散射法对外泌体的粒径进行测定,一式三份。结果显示,外泌体的粒径为67.27±5.0nm,PDI为0.785±0.0160,见图1。
采用冷冻透射电子显微镜(Cryo-TEM)观察外泌体的形态。将5μL外泌体(4mg/mL)样品滴加至亲水化处理的铜网上,等待30s后用滤纸吸干多余水分,随后立即插入液态乙烷中进行冷冻制样,液氮中保存备用。将样品转移入冷冻透射电镜的样品室内。抽真空后,在200kV加速电压下观察并拍摄。Cryo-TEM结果显示观察到的外泌体呈现球形,结构完整,直径主要在30-100nm范围内,见图2。
二、诱导乳腺上皮细胞外泌体促进毛发再生
选取7周龄的C57/BL雌性小鼠,用电动剃须刀剃掉背部毛发,随后用脱毛膏涂抹于背部,等待10秒-20秒后,用纸巾擦去背部毛发露出粉红色皮肤。次日,将溶于PBS中的外泌体进行皮下注射,每次注入250μg外泌体,两天注射一次,连续处理两周。结果发现,随着处理时间的增加,小鼠背部两侧粉红色的皮肤慢慢变黑,随后生长出黑色毛发,毛发不断增多,长满整个背部(见图3)。
注射外泌体两周后,取处理组(注射外泌体)与对照组(注射PBS)小鼠皮肤组织进行切片以及HE染色和Masson染色。结果显示,与注射PBS组相比,注射外泌体组皮肤组织的真皮层更加宽厚,毛囊数目也显著增加(图4),说明诱导乳腺上皮细胞外泌体具有激活毛囊细胞,促进毛囊以及毛发再生的作用。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.一种促进毛发再生的诱导乳腺上皮细胞外泌体,其特征在于:其获得包括诱导乳腺上皮细胞的获得以及诱导乳腺上皮细胞外泌体的提取两个步骤,其中诱导乳腺上皮细胞通过如下方法获得,将成纤维细胞或表皮细胞以1×104~5×106个细胞的密度接种至60mm的培养皿中,高糖DMEM+体积百分比10%FBS培养8-24h后,更换诱导培养基R,然后置于37℃,5%CO2培养箱继续培养8天,其中每间隔两天更换一次培养基;更换培养基时,将上清液进行收集,用于外泌体的提取。
2.根据权利要求1所述的促进毛发再生的诱导乳腺上皮细胞外泌体,其特征在于:所述诱导培养基R,包括Knockout DMEM/F12、N2(100×)、Neurobasal、B27(50×)、Glutamine(100×)以及Repsox(R),其中Knockout DMEM/F12、N2(100×)、Neurobasal、B27(50×)、Glutamine(100×)的体积比为99:1:97:2:1,Repsox(R)的浓度为5-100μM。
3.根据权利要求1所述的促进毛发再生的诱导乳腺上皮细胞外泌体,其特征在于:细胞源于人、小鼠、大鼠、兔、猪、羊、山羊、牛或水牛耳部成纤维细胞或表皮细胞。
4.根据权利要求1所述的促进毛发再生的诱导乳腺上皮细胞外泌体,其特征在于:成纤维细胞通过如下方法获得,将贴壁状态良好的山羊耳边缘组织块加入到DMEM培养液进行贴壁培养,每2d~3d更换一次培养基待;原代培养的单层细胞在培养皿中汇合度达80-90%即可进行传代,弃去旧培养液,质量百分比0.1%~0.3%胰蛋白酶进行消化,然后高糖DMEM+10%FBS(体积百分比)培养基进行中和;收集细胞悬液,离心1200r/min,1~3min后弃上清,重悬细胞后均匀接种。
5.根据权利要求1所述的促进毛发再生的诱导乳腺上皮细胞外泌体,其特征在于:采用试剂盒提取法对诱导乳腺上皮细胞外泌体进行提取。
6.根据权利要求5所述的促进毛发再生的诱导乳腺上皮细胞外泌体,其特征在于:对诱导乳腺上皮细胞外泌体进行提取使用的试剂盒为#41201ES50。
7.根据权利要求6所述的促进毛发再生的诱导乳腺上皮细胞外泌体,其特征在于:对诱导乳腺上皮细胞外泌体进行提取包括如下步骤,将诱导乳腺上皮细胞的获得阶段收集的上清液,3000~5000×g,离心10~15min,收集上清;每10mL细胞培养上清加入2~3mL外泌体抽提试剂,涡旋振荡混匀1~2min,再置于2-8℃静止2~3h;将装有混合液的离心管于4℃,10000×g离心30min~60min,弃上清,收集沉淀;取30~100μL PBS均匀吹打沉淀物,转移至1.5mL离心管中;将含有外泌体的离心管于2-8℃,12000~15000×g离心2~3min,弃沉淀保留上清液,置于-80℃冰箱保存。
8.根据权利要求1-7任一项所述的诱导乳腺上皮细胞外泌体在制备促进毛发再生药物或护理品中的应用。
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