CN114917250A - 成纤维细胞外囊泡hFB-EVs在治疗脱发产品中的应用 - Google Patents
成纤维细胞外囊泡hFB-EVs在治疗脱发产品中的应用 Download PDFInfo
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Abstract
本发明的目的在于提供成纤维细胞衍生的细胞外囊泡hFB‑EVs的新应用。具体是,成纤维细胞衍生的细胞外囊泡hFB‑EVs在治疗脱发产品中的应用。本发明研究了一类特定的细胞外囊泡(EVs),即人类正常成纤维细胞衍生的EVs(hFB‑EVs),对人类真皮乳头(DP)和外根鞘(ORS)细胞的影响,并检查了负责毛囊(HF)中毛发生长的分子机制。本发明发现维持人类真皮乳头细胞生发活性的Wnt3a与hFB‑EVs相比与成纤维细胞相比更丰富且更紧密相关。此外,hFB‑EV相关的Wnt3a介导培养的DP细胞中的受体激活,导致细胞质中β‑连环蛋白的增加及其易位到细胞核中,从而提高靶基因Axin2和Lef1的表达。此外,hFB‑EVs能够促进了人毛囊中外根鞘细胞的迁移、增殖和分化以及毛干的伸长。这些发现揭示了hFB‑EV影响头发生长的新机制。
Description
技术领域
本发明涉及生发技术领域,具体涉及成纤维细胞衍生的细胞外囊泡hFB-EVs在治疗脱发产品中的应用。
背景技术
脱发是一种常见的医学疾病,其特征是不可预测的脱发,影响男性和女性。虽然在大多数情况下,脱发不超过几个斑块,但在少数情况下,它可能更极端(全秃)。所有成熟的毛囊(HF)都会经历一个包含四个阶段的生长周期:生长期(生长)、退行期(退化)、休止期(休息)和外生期(脱落)。毛囊真皮乳头(DP)是一种特殊的成纤维细胞,已知通过调节毛囊中的各种细胞活动在毛发形态发生和循环中发挥关键作用。由外根鞘(ORS)的毛囊干细胞产生的毛基质祖细胞在毛囊以下迅速增殖和迁移,并进一步分化为有丝分裂后基质细胞和内根鞘细胞,从而引起毛干角化脱落,进而导致脱发。
细胞外囊泡(EVs)是由大多数细胞分泌的纳米级膜状囊泡。细胞外囊泡主要分为两种类型:外泌体(50-150nm)和微泡(50-400nm)。外泌体由多泡体通过复杂的机制在细胞内形成,而微泡则在细胞膜中形成。它们被释放到活生物体的体外介质和细胞外空间中,并存在于所有生物体液(尿液、血液、羊水、唾液和母乳)中。
细胞外囊泡可以携带各种生物材料,包括脂质、蛋白质、miRNA、mRNA和DNA等。身体远处的细胞可以通过发出由一种或多种分子组成的信号来交换信息;因此,细胞外囊泡对于非直接接触的细胞之间的长距离通信很重要。
最近,已经进行了广泛的研究以了解细胞外囊泡的生物学和治疗功能,但只有少数研究报告了细胞外囊泡对头发生长的治疗作用。本发明研究证明了在体外诱导人类毛囊真皮乳头细胞增殖和迁移,并通过细胞外囊泡(从小鼠间充质干细胞分泌)促进毛囊从休止期转变为生长期。另一项研究表明,毛囊真皮乳头细胞衍生的外泌体通过增加外根鞘细胞中的β-连环蛋白(β-catenin)和声波刺猬蛋白(sonic hedge hogprotein,SHH)水平来促进增殖和迁移,从而调节毛囊的发展。
因此,本发明旨在研究人类正常成纤维细胞衍生的细胞外囊泡(hFB-EVs)对人类毛囊真皮乳头和外根鞘细胞的影响,并研究导致毛囊细胞毛发生长的分子机制。
发明内容
本发明的目的是提供成纤维细胞衍生的细胞外囊泡hFB-EVs在治疗脱发产品中的应用,其揭示了hFB-EVs能够促进了人毛囊中外根鞘细胞的迁移、增殖和分化以及毛干的伸长。
为解决上述技术问题,本发明采用如下技术方案:
成纤维细胞外囊泡hFB-EVs在治疗脱发产品中的应用。
一种促进毛发生长的产品,包括成纤维细胞衍生的细胞外囊泡hFB-EVs。
本发明的有益效果在于:本发明的结果表明,成纤维细胞衍生的细胞外囊泡(hFB-EVs)在其表面含有Wnt3a可通过激活毛囊真皮乳头细胞中的Wnt/β-catenin信号传导来调节毛发生长,并且hFB-EVs还增加了外根鞘细胞中的分化标志物。基于这些结果,可以得出结论,hFB-EVs具有治疗脱发的潜力。
具体实施方式
下面将结合本发明实施例中对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实验内容:
(1)细胞培养
人成纤维细胞在细胞培养基中培养,添加10%EV去除胎牛血清(超高速离心120000g(4℃,18小时)和1%青霉素-链霉混合液(双抗),温度为37℃,存放在5%CO2培养箱中培养。
(2)免疫荧光
每孔将一千个细胞接种到8孔室载玻片中并孵育过夜。附着在载玻片上后,用4%多聚甲醛固定载玻片中的细胞。PBS中的BSA(3%)用于封闭载玻片中的细胞。随后用抗αSMA或抗Wnta3抗体探测它们过夜,用PBS洗涤(3次),并用二级AlexaFluor荧光染料进一步探测555抗体1小时。细胞用PBS洗涤(三次)并使用含有DAPI的封固剂封固。用蔡司超分辨率共聚焦显微镜对载玻片中的细胞进行成像。
(3)从人头皮中分离和培养毛囊真皮乳头细胞和外根鞘细胞
从头皮皮肤获得生长期的毛囊。毛囊真皮乳头从解剖的毛囊球茎中分离出来,转移到涂有牛I型胶原蛋白的塑料培养皿上,并在补充有1%抗生素抗真菌溶液和20%热灭活胎牛血清(FBS)在37℃含有5%CO2的环境中。这些外植体每3天更换一次培养基。达到汇合后,用PBS中的0.25%胰蛋白酶/10mmEDTA收获细胞,以1:4的比例分裂,然后维持在10%FBS补充的细胞培养基中。
外根鞘细胞是从与毛囊真皮乳头细胞相同的毛发样本中分离出来的。去除毛囊中的毛干和毛球区域以防止与其他细胞的污染。将修剪过的毛囊浸入BiocoatI型胶原蛋白包被的组织培养皿中的20%FBS补充培养基中。培养3天后,将培养基更换为角质形成细胞生长培养基,含有1%抗生素抗真菌溶液和1%EpiLife培养基作为生长补充剂。达到汇合后,用PBS中的0.25%胰蛋白酶/10mmEDTA收获细胞,以1:5的比例分离,并维持在EpiLife培养基中。来自第二代的细胞用于进一步的实验。
(4)细胞外囊泡的分离和提取
成纤维细胞在FBS(EVs耗尽)中生长,并通过在4℃下以120000g离心18小时后进行收获,然后通过差速离心分离细胞外囊泡,通过连续离心从细胞培养基中去除细胞碎片。随后,无细胞上清液通过0.45-μm过滤器过滤,然后以100000g超速离心60分钟。在此之后,进行了两步碘克沙醇密度梯度超速离心。细胞外囊泡是从60%和20%碘克沙醇层的交叉点获得的,并储存在-80℃冰箱内保存。将颗粒重新悬浮在50-100μLPBS中并立即使用。所有超速离心步骤均使用超速离心机在4℃下使用Ultra-Clear管进行。
(5)透射电子显微镜观察
进行透射电子显微镜(TEM)观察,将成纤维细胞衍生的细胞外囊泡(hFB-EV)颗粒在4℃下重悬于2%多聚甲醛中过夜;将10μL的悬浮液置于Formvar碳涂层EM网格表面上20分钟。随后用100μL的PBS液滴清洗hFB-EV,将其移液到干净的封口膜上。使用干净的镊子将网格倒置并放置在PBS上。随后将网格与50μL的1%戊二醛液滴一起孵育5分钟。然后用2%乙酸双氧铀对网格进行负染色5分钟,然后用蒸馏水洗涤(7次,每次2分钟)。使用透射电子显微镜观察细胞外囊泡。
(6)蛋白质印迹检测
加载等量的蛋白质并通过10%SDS/PAGE分离。将蛋白质转移到PVDF膜,首先用一抗[(Wnt3a蛋白),(β-肌动蛋白,细胞信号传导),(β-连环蛋白,细胞信号传导),(组蛋白H3,细胞信号)],然后使用与辣根过氧化物酶((Horse Radish Peroxidase,HRP))偶联的二抗,使用增强化学发光检测信号。使用Picasa3(版本3.9.1.4.1)和/或PowerPoint程序裁剪和准备印迹图像,使用GelQuant测量条带强度。
(7)流式细胞仪鉴定
进行细胞外囊泡的流式细胞术分析。通过混合5μg成纤维细胞衍生的细胞外囊泡(hFB-EV),使hFB-EV附着在4μm醛/硫酸盐乳胶珠上,并在室温下在旋转振荡器中孵育2小时。通过加入100mm甘氨酸和2%BSA终止反应,并在室温下在旋转振荡器中孵育30分钟。将10μLWnt3a抗体混合物在4℃孵育过夜,然后在37℃孵育Alexa Fluor FITC荧光标记染料兔多抗体60分钟。用PBS中的2%BSA将样品稀释至1mL,并以15000g离心2分钟。弃去上清液,将珠子重新悬浮在1mLPBS中,使用BDFACS Aria III仪器进行流式细胞术分析。
(8)hFB-EV的相互作用和内化
为了分析成纤维细胞衍生的细胞外囊泡(hFB-EV)相互作用和内化,将5或10μg·mL-1hFB-EV用1,1`-双十八烷基-3,3,3`,3`-四甲基吲哚二碳花菁高氯酸盐,在8孔室中生长的毛囊真皮乳头或外根鞘细胞与细胞膜荧光探针DID标记液标记的hFB-EV在37℃和5%CO2环境中孵育1小时和3小时,固定在甲醇中。用3%的BSA封闭腔室30分钟。将细胞与PBS或Caveolin1一抗(稀释度-1:200)在4℃下孵育过夜。将小室用PBS(3次)洗涤,并与兔用抗山羊抗体和FITC(稀释度-1:200)一起孵育1小时。然后,用PBS洗涤室(三次)。使用含有DAPI染色试剂的安装介质安装载玻片。使用蔡司超分辨率共聚焦显微镜获取图像。
(9)细胞增殖试验
在100μL完全培养基中,用0、1、2、3、4和5μg·mL-1成纤维细胞衍生的细胞外囊泡(hFB-EV)和10μmXAV939抑制剂接种毛囊真皮乳头细胞或外根鞘细胞(n=5000个细胞/孔)一式三份装入96孔微量滴定板中。使用CCK8测定法,在24小时测量增殖。24h时,每孔加入10μLCCK8溶液。将板在CO2培养箱中于37℃下培养4小时。随后,在酶联免疫吸附测定板读数器上测量光密度(OD)。
(10)Transwell细胞迁移试验
Transwell迁移在24孔细胞培养插入物中进行,该插入物含有具有8.0毫米孔的透明PET膜;将外根鞘细胞以1×103个细胞/孔的浓度接种到含有0.5mL无血清培养基以及0、2.5或5μg·mL-1hFB-EV的上室中,并生长24小时。将补充有10%FBS的培养基置于下室并作为化学引诱剂。24小时后,膜下表面的细胞被固定,用结晶紫染色,可视化,进行细胞计数。
(11)实时聚合酶链反应
使用TRIzol试剂溶液裂解细胞,提取总RNA,使用Sso Advanced Universal SYBRGreen Supermix在CFX96touch-Real-timePCR中进行实时PCR。
(12)人发干伸长率
分离和培养人类毛囊。毛囊用不同浓度的成纤维细胞衍生的细胞外囊泡(hFB-EV)(0、0.1、0.5、1μg·mL-1)处理,随后在第3天和第6天测量毛干伸长率。实验使用从两个人获得的13个毛囊进行。
(13)统计分析
所有数据均表示为平均值±标准差(SD)。使用MSOffice或graphpadprism7软件v.7.04使用学生t检验或双向ANOVA方差分析对组之间的差异进行统计分析。P值<0.05被认为具有统计学意义。
本发明研究了一类特定的细胞外囊泡(EVs),即人类正常成纤维细胞衍生的EVs(hFB-EVs),对人类真皮乳头(DP)和外根鞘(ORS)细胞的影响,并检查了负责毛囊(HF)中毛发生长的分子机制。本发明发现维持人类真皮乳头细胞生发活性的Wnt3a与hFB-EVs相比与成纤维细胞相比更丰富且更紧密相关。此外,hFB-EV相关的Wnt3a介导培养的DP细胞中的受体激活,导致细胞质中β-连环蛋白的增加及其易位到细胞核中,从而提高靶基因Axin2和Lef1的表达。此外,hFB-EVs能够促进了人毛囊中外根鞘细胞的迁移、增殖和分化以及毛干的伸长。这些发现揭示了hFB-EV影响头发生长的新机制。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (1)
1.成纤维细胞外囊泡hFB-EVs在治疗脱发产品中的应用。
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