CN113876933A - 一种促毛发生长组合物、制备方法及其应用 - Google Patents
一种促毛发生长组合物、制备方法及其应用 Download PDFInfo
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- CN113876933A CN113876933A CN202111487599.2A CN202111487599A CN113876933A CN 113876933 A CN113876933 A CN 113876933A CN 202111487599 A CN202111487599 A CN 202111487599A CN 113876933 A CN113876933 A CN 113876933A
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Abstract
本发明提供一种促毛发生长的组合物,所述组合物的活性成分包括Laminin‑511修饰的间充质干细胞培养上清液、FGF10修饰的间充质干细胞培养上清液以及Wnt‑10B修饰的间充质干细胞培养上清液,体积比为1:1:1;本发明还提供上述组合物的制备方法和应用。本发明制备的组合物可以促进毛囊的生长和发育,从而促进毛发生长,可有效解决脱发问题,并且治疗效果高达95%;本发明提供的产品使用天然提取物,不含任何非天然添加剂,安全性较高,无副作用;本发明提供的产品中的间充质干细胞培养上清液含有多种细胞因子,可以让患者有效的预防脱发,促进毛发再生。
Description
技术领域
本发明涉及一种促毛发生长精油、制备方法及其应用,属于生物医药技术领域。
背景技术
临床上常见的脱发性疾病有斑秃(alopecia areata)、雄激素性脱发(androgenicalopecia)及生理性脱发(physiological alopecia)。多年来,人们一直在寻求一种安全、可靠的方法来治疗和预防脱发,但很难找到一种安全有效的方法来解决这个问题。目前使用的治疗方法主要是药物治疗,如米诺地尔或普鲁卡因、非那雄胺等, 其中米诺地儿具有刺激毛发生长的效果,而非那雄胺能够抑制5α还原酶的产生,减少DHT对头顶毛囊的再影响,只是这些传统方法见效慢,也无法根本性地解决脱发问题,并且有潜在风险,不适合长期使用。
毛发生长开始于毛囊内,毛囊是一个微小的、高度再生的器官,位于哺乳动物皮肤的真皮层,其中包含大量间充质干细胞,用于在毛发脱落后再生,以及在皮肤受伤后再生。毛囊的正常发育和循环是通过毛囊上皮与间充质真皮乳头的相互作用而发生的,真皮乳头可分泌成纤维细胞生长因子10(FGF10)。近来有研究表明FGF10能促进毛发生长,FGF10的受体FGFR2b特异表达在毛囊外毛根鞘,与毛囊干细胞的增值密切相关,而毛囊干细胞对毛发再生和皮肤烧伤后的修复有重要意义。也有研究表明Wnt 信号在毛囊的生长和发育中也起着关键作用, 在信号分子中,Wnt10b被证明可以通过 Wnt/β-Catenin信号通路促进毛囊的生长和真皮乳头细胞的增殖,可以促进原代皮肤上皮细胞向毛囊细胞的毛干和内根鞘分化。
Laminin-511 是由三种不同的糖基化多肽链组成,称为 α、β 和 γ,它们组装成二硫键连接的三聚体。通过微针阵列装置及其用于局部(包括皮肤)向受试者施用层粘连蛋白-511肽或蛋白质也可以增加头皮毛发生长。
中国专利CN201110198170.1:中药育发液,多种药材混匀,得到混合药材;将混合药材与60vol%乙醇水溶液按质量比1∶1混合,在密闭条件下放置,使药材充分湿润和膨胀,得到膨胀的药料;再对膨胀的药料进行渗漉,得到的渗漉液即为中药育发液。
中国专利CN201010522342.1:属于化妆品领域,该外用育发液是由生发剂、保湿剂、香味剂、防腐剂经适量溶剂调配而成。
中国专利CN201910598631.0 :提供了一种用于修复毛囊的育发液制剂,所述育发液制剂包括占巴花提取液、干细胞提取物溶液、防腐剂、肌醇、黄原胶、去离子水和pH调节剂。该育发液中含有干细胞提取物溶液为间充质干细胞破碎细胞液或者间充质干细胞培养液。
目前使用的治疗方法主要是药物治疗,如米诺地尔或普鲁卡因、非那雄胺等, 其中米诺地儿具有刺激毛发生长的效果,而非那雄胺能够抑制5α还原酶的产生,减少DHT对头顶毛囊的再影响,只是这些传统方法见效慢,也无法根本性地解决脱发问题,并且有潜在风险,不适合长期使用。
育发的天然植物制成产品种类繁多,目前的天然植物类育发产品多数是采用大量药材组成复杂配方,如多种中药配方的中国专利CN201110198170.1 ;一种植物性化妆品组合物中国专利CN201010522342.1。这些育发产品发挥的作用不明确,难以发挥育发某个植物的最大疗效,治愈率低。
中国专利CN201910598631.0 :提供了一种用于修复毛囊的育发液制剂,含有干细胞提取物溶液为间充质干细胞破碎细胞液或者间充质干细胞培养液,虽然也能作用于毛囊组织来修复毛囊,但治疗效果不佳。
发明内容
针对现有技术存在的不足,本发明提供一种促毛发生长组合物、制备方法及其应用,实现以下发明目的:修复毛囊,促进毛发生长。
为解决上述技术问题,本发明采取以下技术方案:
一种促毛发生长的组合物,所述组合物的活性成分包括Laminin-511修饰的间充质干细胞培养上清液、FGF10修饰的间充质干细胞培养上清液以及Wnt-10B修饰的间充质干细胞培养上清液,体积比为1:1:1。
以下是对上述技术方案的进一步改进:
所述Laminin-511修饰的间充质干细胞培养上清液中,Laminin-511蛋白的浓度为608ng/mL;所述FGF10修饰的间充质干细胞培养上清液中FGF10蛋白的浓度为771ng/mL;所述Wnt-10B修饰的间充质干细胞培养上清液中Wnt-10B蛋白的浓度为666 ng/mL。
所述Laminin-511修饰的间充质干细胞,采用Laminin-511质粒转染间充质干细胞;所述Laminin-511质粒包括Laminin-5α质粒和Laminin-(β+γ)质粒;所述FGF10修饰的间充质干细胞,采用FGF10质粒转染间充质干细胞;所述Wnt-10B修饰的间充质干细胞,采用Wnt-10B质粒转染间充质干细胞。
所述Laminin-5α的核酸人工序列为序列表中SEQ ID NO.2所示;所述Laminin-(β+γ),包括1β核酸人工序列、1γ核酸人工序列;所述1β核酸人工序列为序列表中SEQ IDNO.3所示,所述1γ核酸人工序列为序列表中SEQ ID NO.4所示;所述FGF10 的核酸人工序列为序列表中SEQ ID NO.5所示,所述Wnt-10B核酸人工序列为SEQ ID NO.6所示。
所述组合物中还包括50μg/mL的乳铁转运蛋白、50μg/mL的生物素、8μg/mL的维生素E、6μg/mL的维生素B5、1μg/mL的地高辛。
一种促毛发生长的组合物的制备方法,将所述的活性成分按照体积比为1:1:1混合,加入乳铁转运蛋白、生物素、维生素E、维生素B5、地高辛,混合均匀得到组合物。
一种促毛发生长的组合物的应用,所述的组合物用于促进毛囊和头发生长,抑制脱发。
与现有技术相比,本发明取得以下有益效果:
(1)本发明制备的组合物可以促进毛囊的生长和发育,从而促进毛发生长,可有效解决脱发问题,并且治疗效果高达95%。
(2)本发明提供的产品使用天然提取物,不含任何非天然添加剂,安全性较高,无副作用。
(3)本发明提供的产品中的间充质干细胞培养上清液含有多种细胞因子,比如:成纤维细胞生长因子(FGF)、内皮细胞生长因子(VEGF)、表皮细胞生长因子(EGF)、白细胞介素(IL)、肿瘤坏死因子-α (TNF-α)等能有效的抑制IFNG,CXCL9和CXCL10的产生以及NKG2D+T细胞的渗透,可以让患者有效的预防脱发,促进毛发再生。
(4)本发明采用Laminin-511修饰间充质干细胞、 FGF10修饰间充质干细胞、Wnt-10B修饰间充质干细胞,通过对核苷酸序列进行优化,可以提高 Laminin-511、FGF10、Wnt-10B蛋白的分泌量。
附图说明
图1为P4代的脐带间充质干细胞;
图2为含目的质粒的腺病毒感染间充质干细胞后的GFP表达率;
图3为间充质干细胞中Laminin-511、FGF10以及Wnt-10B蛋白的表达量的柱状图;
图4为本发明组合物和对照组体外毛囊生长的对比图。
具体实施方式
下面结合具体的实施例对本发明进一步说明。但这些例举性实施方式的用途和目的仅用来例举本发明,并非对本发明的实际保护范围构成任何形式的任何限定,更非将本发明的保护范围局限于此。
实施例1
构建表达载体:分别是Laminin-511质粒(包括Laminin-5α质粒和Laminin-(β+γ)质粒)、FGF10质粒、Wnt-10B质粒;
Laminin-5α,包括signal核酸人工序列(SEQ ID NO.1)和 5α核酸人工序列(SEQID NO.2),分别按SEQ ID NO.1和SEQ ID NO.2的核酸编码序列委托生工生物工程(上海)有限公司合成其整个表达框,插入到腺病毒表达载体pAAV-IRES-ZsGreen1 的BamHI-EcoRI位点,转化到E.coli(DH5α),经测序正确后,使用Invitrogen公司的质粒纯化试剂盒提取并纯化质粒,获得重组表达载体的高品质质粒。
Laminin-(β+γ),包括signal核酸人工序列(SEQ ID NO.1),1β核酸人工序列(SEQID NO.3),1γ核酸人工序列(SEQ ID NO.4)分别按SEQ ID NO.1、SEQ ID NO.3和SEQ IDNO.4的核酸编码序列委托生工生物工程(上海)有限公司合成其整个表达框,插入到腺病毒表达载体pAAV-IRES-ZsGreen1 的BamHI-EcoRI位点,转化到E.coli(DH5α),经测序正确后,使用Invitrogen公司的质粒纯化试剂盒提取并纯化质粒,获得重组表达载体的高品质质粒。
FGF10质粒,包括signal核酸人工序列(SEQ ID NO.1)和FGF10 核酸人工序列(SEQID NO.5),分别按SEQ ID NO.1和SEQ ID NO.5的核酸编码序列委托生工生物工程(上海)有限公司合成其整个表达框,插入到腺病毒表达载体pAAV-IRES-ZsGreen1 的BamHI-EcoRI位点,转化到E.coli(DH5α),经测序正确后,使用Invitrogen公司的质粒纯化试剂盒提取并纯化质粒,获得重组表达载体的高品质质粒。
Wnt-10B质粒,包括signal核酸人工序列(SEQ ID NO.1)和 Wnt-10B核酸人工序列(SEQ ID NO.6),分别按SEQ ID NO.1和SEQ ID NO.6的核酸编码序列委托生工生物工程(上海)有限公司合成其整个表达框,插入到腺病毒表达载体pAAV-IRES-ZsGreen1 的BamHI-EcoRI位点,转化到E.coli(DH5α),经测序正确后,使用Invitrogen公司的质粒纯化试剂盒提取并纯化质粒,获得重组表达载体的高品质质粒。
将上述四种质粒分别转化到E.coli(DH5α)。筛选鉴定出阳性克隆,选取阳性克隆37℃、250rpm摇菌16h,按照质粒提取纯化试剂盒(购自Invitrogen公司)提取Laminin-5α、Laminin-(β+γ)、FGF10质粒、Wnt-10B质粒,具体步骤见说明书。将上述的4种质粒委托生工生物工程(上海)有限公司进行测序。经测序正确后备用,将上述提取的四种质粒均稀释至1μg/μL。
实施例2
脐带间充质干细胞的制备
取医院捐献的新生儿脐带,在超净工作台中先用75%酒精消毒两次,放在培养皿中,用镊子剥离出其中的华氏胶组织,用剪刀剪碎至0.5mm2 大小的小块。将剪碎的华氏胶组织转移到培养瓶中,加入含有10Vol%血浆替代物的 UltraCULTURE培养基培养,每天用显微镜观察。爬出的干细胞铺满瓶底的80%时,进行传代,传代后细胞生长速度加快,每2-3天传一代,传至P4代用于实验(见图1)。
实施例3腺病毒包装及滴度检测
(一)293T细胞的复苏
从液氮罐中取出冻存的293T细胞,迅速丢入37℃水浴锅中并快速晃动,直至细胞溶液完全溶解。
将1mL浓度为106个/mL细胞溶液转移到50mL离心管中,并在其中加上10 mL新鲜的完全培养基,混匀后离心,1500 rpm,5 min。
去掉上清,加入3 mL新鲜的完全培养基进行重悬细胞,平均转入六孔板中,每个孔补足到3mL培养基;
将六孔板平稳放入37 ℃、5 %CO2 和95 %相对湿度的培养箱中培养。
第二天观察细胞存活率,并更换新鲜的完全培养基。
以后每天观察细胞生长情况,细胞铺满孔底时,进行传代,传代10次后,待长满80%瓶底时用于实验。
所述完全培养基为含有10Vol%FBS的DMEM培养基。
(二)腺病毒包装与滴度测定
(1)腺病毒包装Laminin-511质粒
取两个1.5mL无菌 EP管,一个EP管中加入200µL无血清DMEM,加入2µg辅助质粒和1µg Laminin5α;另一个EP管中加入200µl无血清DMEM,加入6µl lipoLP2000,静止5min。然后将两管充分混匀,静置20min,得到Laminin5α包装液;
Laminin-(β+γ)质粒替换Laminin5α质粒,重复上述操作,得到Laminin-(β+γ)包装液;
将上述Laminin5α包装液、Laminin-(β+γ)包装液,混合后逐滴加入上述长满80%细胞的6孔板中,轻轻摇动混匀后置于含37℃恒温箱孵育;6h后吸去培养基,加入适量37℃预热的DMEM完全培养基,继续将细胞放置37℃恒温箱孵育。
(2)腺病毒包装FGF10质粒
取两个1.5mL无菌 EP管,一个EP管中加入200µl无血清DMEM,加入2µg辅助质粒和1µgFGF10质粒;另一个EP管中加入200µl无血清DMEM,加入6µl lipoLP2000,静止5min。然后将两管充分混匀,静置20min;将混合液逐滴加入上述长满80%细胞的6孔板中,轻轻摇动混匀后置于含37℃恒温箱孵育;6h后吸去培养基,加入适量37℃预热的DMEM完全培养基,继续将细胞放置37℃恒温箱孵育。
(3)腺病毒包装Wnt-10B质粒
取两个1.5mL无菌 EP管,一个EP管中加入200µl无血清DMEM,加入2µg辅助质粒和1µg Wnt-10B质粒;另一个EP管中加入200µl无血清DMEM,加入6µl lipoLP2000,静止5min。然后将两管充分混匀,静置20min;将混合液逐滴加入上述长满80%细胞的6孔板中,轻轻摇动混匀后置于含37℃恒温箱孵育;6h后吸去培养基,加入适量37℃预热的DMEM完全培养基,继续将细胞放置37℃恒温箱孵育。
(4)滴度测定
转染48h后收集含腺病毒的上清,用0.45μm小滤器过滤,收集滤液后用流式细胞仪进行滴度测定。根据公式:滴度(pfu/mL)=细胞数×荧光百分比×MOI(1)×病毒稀释倍数×103 计算病毒滴度,本发明中获得的腺病毒病毒液的滴度分别为3.36×107pfu/mL(Laminin-511)、3.52×107 pfu/mL(FGF10)、3.4×107 pfu/mL(Wnt-10B)。
实施例4腺病毒感染间充质干细胞
将生长状态良好的间充质干细胞(实施例2得到的p4代的间充质干细胞)消化计数后稀释至密度为1×105个细胞/mL,加入六孔板中,每孔加入量为3 mL,置于37℃、5%CO2培养箱中培养24h。
从-80℃分别取出实施例3获得3种腺病毒病毒液(2mL),加入终浓度为8μg/mL的聚凝胺(购自Sigma公司),得到含聚凝胺的病毒液。
从培养箱中拿出六孔板,去掉六孔板中的培养基,加入准备好的含聚凝胺的病毒液,放入培养箱中继续培养24h。24h后,去掉六孔板中的病毒液,添加完全培养基(含有10Vol %FBS、1 Vol %青链霉素的H-DMEM培养基,均购自Gibco公司)进行培养(每孔加入3 mL培养基),同时加入3μg/mL的嘌呤霉素进行筛选,放在37℃,5% CO2 培养箱中培养,每2天换液一次,培养5天(从加入病毒液开始算),成活的细胞即为腺病毒感染的间充质干细胞。流式细胞仪检测分别间充质干细胞GFP表达率,如图2 所示,本发明中Laminin-511质粒病毒感染间充质干细胞GFP表达率为27%,FGF10质粒病毒感染间充质干细胞GFP表达率为29.3%;Wnt-10B质粒病毒感染间充质干细胞GFP表达率为27%。
实施例5
ELISA检测间充质干细胞中Laminin-511、FGF10以及Wnt-10B蛋白的表达量
样本处理:分别将实施例4中3种腺病毒感染的间充质干细胞传代,用间充质干细胞培养基(含有10Vol%血浆替代物的 UltraCULTURE培养基培养)继续培养,分别收集这3种病毒感染后的第一代细胞、第三代细胞、第五代细胞、 第七代细胞的培养基作为实验组,未转染间充质干细胞的培养基作为阴性对照。离心后取上清溶液分别进行ELISA检测Laminin-511、FGF10以及Wnt-10B蛋白的表达量(如图3)。
结果如图3所示,感染病毒的间充质干细胞其Laminin-511、FGF10以及Wnt-10B蛋白表达量明显高于正常的未转染间充质干细胞的表达量,并且随着细胞传代增值,Laminin-511、FGF10以及Wnt-10B蛋白持续表达,且表达量稳定,没有很大变化。
Laminin-511修饰的第三代间充质干细胞培养上清液中Laminin-511蛋白的浓度为608ng/mL;
FGF10修饰的第三代间充质干细胞培养上清液中FGF10蛋白的浓度为771ng/mL;
Wnt-10B修饰的第三代间充质干细胞培养上清液中Wnt-10B蛋白的浓度为666 ng/mL。
实施例6
一种促毛发生长的精油
所述精油包括活性成分: Laminin-511、FGF10以及Wnt-10B修饰的间充质干细胞培养上清液。
所述精油还包括下述组分:
表1
所述Laminin-511修饰的间充质干细胞培养上清液,即转染Laminin-511质粒的第三代间充质干细胞,并表达Laminin-511蛋白的间充质干细胞培养上清液。
所述FGF10修饰的间充质干细胞培养上清液,即转染FGF10质粒的第三代间充质干细胞,并表达FGF10蛋白的间充质干细胞培养上清液。
所述Wnt-10B修饰的间充质干细胞培养上清液,即转染Wnt-10B质粒的第三代间充质干细胞,并表达Wnt-10B蛋白的间充质干细胞培养上清液。
将Laminin-511、FGF10或Wnt-10B修饰的间充质干细胞培养上清液,按照体积比(1:1:1)混合,其他成分按照上表所示的终浓度加入到上述间充质干细胞培养上清液中,混合均匀,得到精油。
实施例7 精油促进兔须的毛囊生长
分离6周左右兔子的兔须的毛囊,将分离得到的兔须毛囊随机分为两组:实验组和对照组,分别放在细胞培养皿中(5根),实验组用实施例6得到的精油培养,精油的加入体积为10mL,对照组用基础培养基培养,每天更换一次培养基, 5 天后,分别观察两组兔须毛囊的轴生长。其中基础培养基为未转染间充质干细胞培养上清液与实施例6中的其他成分的混合物。
培养条件为置于37℃、5% CO2培养箱中培养。
如图4所示,与本产品精油共培养的兔须毛囊比在基础培养基中培养的毛囊生长得更快,故本产品精油能够促进体外毛囊生长。
实施例8头发生长情况评价
对本产品精油进行活性试验,以观察效果。
实验人数为40个人,均有病理性脱发症状,年龄:18-45岁,随机分为两组:实验组和对照组。其中实验组使用本产品精油,对照组使用实施例6所述的基础培养基。
使用方法:受试前需要实验对象用清水洗净头部并吹干头发,经生发精华或者基础培养基均匀喷于发根,每天两次(早晚),每次喷1mL(喷5-6次),按摩几分钟。所有实验对象用药后,进行30分钟的观察,观察是否有任何可能的急性过敏反应的迹象,并且两组治疗2个月后随访,对比两组治疗效果。
实验结果:
疗效标准分为:(1)显效:无明显脱发,有大量新毛发生成;(2)有效:脱发得到控制,有少量新毛发生成;(3)无效:治疗前后无变化。
本产品精油疗效结果如表2所示:
由表2结果表明:本产品精油治疗效果为95%,明显高于对照组的治疗效果,且具有统计学意义。
序列表
<110> 山东兴瑞生物科技有限公司
<120> 一种促毛发生长组合物、制备方法及其应用
<141> 2021-11-18
<160> 6
<170> SIPOSequenceListing 1.0
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<212> DNA
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gccctggaag aagccatgct gcaggagcag cagcgtctgg gtctggtatg ggctgcactg 180
cagggtgctc gtactcaact ccgagatgtc cgggccaaga aggaccagct ggaggcgcac 240
atccaggcgg cgcaggccat gcttgccatg gacacagacg agacaagcaa gaagatcgca 300
catgccaagg ctgtggctgc tgaagcccag gacaccgcca cccgtgtgca gtcccagctg 360
caggccatgc aggagaatgt ggagcggtgg cagggccagt acgagggcct gcggggccag 420
gacctgggcc aggcagtgct tgacgcaggc cactcagtgt ccaccctgga gaagacgctg 480
ccccagctgc tggccaagct gagcatcctg gagaaccgtg gggtgcacaa cgccagcctg 540
gccctgtccg ccagcattgg ccgcgtgcga gagctcattg cccaggcccg gggggctgcc 600
agtaaggtca aggtgcccat gaagttcaac gggcgctcag gggtgcagct gcgcacccca 660
cgggatcttg ccgaccttgc tgcctacact gccctcaagt tctacctgca gggcccagag 720
cctgagcctg ggcagggtac cgaggatcgc tttgtgatgt acatgggcag ccgccaggcc 780
actggggact acatgggtgt gtctctgcgt gacaagaagg tgcactgggt gtatcagctg 840
ggtgaggcgg gccctgcagt cctaagcatc gatgaggaca ttggggagca gttcgcagct 900
gtcagcctgg acaggactct ccagtttggc cacatgtccg tcacagtgga gagacagatg 960
atccaggaaa ccaagggtga cacggtggcc cctggggcag aggggctgct caacctgcgg 1020
ccagacgact tcgtcttcta cgtcgggggg taccccagta ccttcacgcc ccctcccctg 1080
cttcgcttcc ccggctaccg gggctgcatc gagatggaca cgctgaatga ggaggtggtc 1140
agcctctaca acttcgagag gaccttccag ctggacacgg ctgtggacag gccttgtgcc 1200
cgctccaagt cgaccgggga cccgtggctc acggacggct cctacctgga cggcaccggc 1260
ttcgcccgca tcagcttcga cagtcagatc agcaccacca agcgcttcga gcaggagctg 1320
cggctcgtgt cctacagcgg ggtgctcttc ttcctgaagc agcagagcca gttcctgtgc 1380
ttggccgtgc aagaaggcag cctcgtgctg ttgtatgact ttggggctgg cctgaaaaag 1440
gccgtcccac tgcagccccc accgcccctg acctcggcca gcaaggcgat ccaggtgttc 1500
ctgctggggg gcagccgcaa gcgtgtgctg gtgcgtgtgg agcgggccac ggtgtacagc 1560
gtggagcagg acaatgatct ggagctggcc gacgcctact acctgggggg cgtgccgccc 1620
gaccagctgc ccccgagcct gcgacggctc ttccccaccg gaggctcagt ccgtggctgc 1680
gtcaaaggca tcaaggccct gggcaagtat gtggacctca agcggctgaa cacgacaggc 1740
gtgagcgccg gctgcaccgc cgacctgctg gtggggcgcg ccatgacttt ccatggccac 1800
ggcttccttc gcctggcgct ctcgaacgtg gcaccgctca ctggcaacgt ctactccggc 1860
ttcggcttcc acagcgccca ggacagtgcc ctgctctact accgggcgtc cccggatggg 1920
ctatgccagg tgtccctgca gcagggccgt gtgagcctac agctcctgag gactgaagtg 1980
aaaactcaag cgggcttcgc cgatggtgcc ccccattacg tcgccttcta cagcaatgcc 2040
acgggagtct ggctgtatgt cgatgaccag ctccagcaga tgaagcccca ccggggacca 2100
ccccccgagc tccagccgca gcctgagggg cccccgaggc tcctcctggg aggcctgcct 2160
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ctgggcccac agcgcgtatt tgatctgcag cagaacctgg gcagcgtcaa tgtgagcacg 2280
ggctgtgcac ccgccctgca agcccagacc ccgggcctgg ggcctagagg actgcaggcc 2340
accgcccgga aggcctcccg ccgcagccgt cagcccgccc ggcatcctgc ctgcatgctg 2400
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gaagcagaaa aggcccaggt cgcagcagag aaggcaatta aacaagcaga tgaagacatt 180
caaggaaccc agaacctgtt aacttcgatt gagtctgaaa cagcagcttc tgaggaaacc 240
ttgttcaacg cgtcccagcg catcagcgag ttagagagga atgtggaaga acttaagcgg 300
aaagctgccc aaaactccgg ggaggcagaa tatattgaaa aagtagtata tactgtgaag 360
caaagtgcag aagatgttaa gaagacttta gatggtgaac ttgatgaaaa gtataaaaaa 420
gtagaaaatt taattgccaa aaaaactgaa gagtcagctg atgccagaag gaaagccgaa 480
atgctacaaa atgaagcaaa aactctttta gctcaagcaa atagcaagct gcaactgctc 540
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aagaccagag aagcccagca ggccctgggc agtgctgcgg cggatgccac agaggccaag 180
aacaaggccc atgaggcgga gaggatcgcg agcgctgtcc aaaagaatgc caccagcacc 240
aaggcagaag ctgaaagaac ttttgcagaa gttacagatc tggataatga ggtgaacaat 300
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caggacatga tgatggcagg gatggcttca caggctgctc aagaagccga gatcaatgcc 420
agaaaagcca aaaactctgt tactagcctc ctcagcatta ttaatgacct cttggagcag 480
ctggggcagc tggatacagt ggacctgaat aagctaaacg agattgaagg caccctaaac 540
aaagccaaag atgaaatgaa ggtcagcgat cttgatagga aagtgtctga cctggagaat 600
gaagccaaga agcaggaggc tgccatcatg gactataacc gagatatcga ggagatcatg 660
aaggacattc gcaatctgga ggacatcagg aagaccttac catctggctg cttcaacacc 720
ccgtccattg aaaagccc 738
<210> 5
<211> 624
<212> DNA
<213> Human sapiens
<400> 5
atgtggaaat ggattctgac ccactgtgca tctgcattcc cgcacctgcc gggttgttgc 60
tgctgctgct tcctgctgct gttcctggtt tcttctgtcc cggttacctg ccaggcgctg 120
ggtcaggaca tggtgagccc ggaagctacc aacagctctt cttcttcctt tagctccccg 180
agctccgctg gtcgccatgt acgttcttat aaccacctgc agggcgacgt acgctggcgt 240
aagctattct ctttcaccaa gtactttctc aagattgaga agaacgggaa ggtcagcggg 300
accaagaagg agaactgccc gtacagcatc ctggagataa catcagtaga aatcggagtt 360
gttgccgtca aagccattaa cagcaactat tacttagcca tgaacaagaa ggggaaactc 420
tatggctcaa aagaatttaa caatgactgt aagctgaagg agaggataga ggaaaatgga 480
tacaatacct atgcatcatt taactggcag cataatggga ggcaaatgta tgtggcattg 540
aatggaaaag gagctccaag gagaggacag aaaacacgaa ggaaaaacac ctctgctcac 600
tttcttccaa tggtggtaca ctca 624
<210> 6
<211> 1167
<212> DNA
<213> Human sapiens
<400> 6
atgctggagg agccccggcc gcggcctccg ccctcgggcc tcgcgggtct cctgttcctg 60
gcgttgtgca gtcgggctct aagcaatgag attctgggcc tgaagttgcc tggcgagccg 120
ccgctgacgg ccaacaccgt gtgcttgacg ctgtccggcc tgtctaagcg tcaactgggt 180
ctgtgtctgc gtaacccgga cgttactgca tctgcgctgc agggtctgca catcgccgtg 240
catgaatgtc agcaccagct gcgtgatcaa cgttggaact gctctgctct ggagggcggt 300
ggtcgtctgc cacatcacag cgccatcctc aagcgcggtt tccgagaaag tgctttttcc 360
ttctccatgc tggctgctgg ggtcatgcac gcagtagcca cggcctgcag cctgggcaag 420
ctggtgagct gtggctgtgg ctggaagggc agtggtgagc aggatcggct gagggccaaa 480
ctgctgcagc tgcaggctct gagccgtggt aaatcctttc cgcattctct gccatctcct 540
ggcccaggtt cttctccttc cccgggtcca caggatacct gggaatgggg cggttgcaac 600
catgacatgg actttggaga gaagttctct cgggatttct tggattccag ggaagctccc 660
cgggacatcc aggcacgaat gcgaatccac aacaacaggg tggggcgcca ggtggtaact 720
gaaaacctga agcggaaatg caagtgtcat ggcacatcag gcagctgcca gttcaagaca 780
tgctggaggg cggccccaga gttccgggca gtgggggcgg cgttgaggga gcggctgggc 840
cgggccatct tcattgatac ccacaaccgc aattctggag ccttccagcc ccgtctgcgt 900
ccccgtcgcc tctcaggaga gctggtctac tttgagaagt ctcctgactt ctgtgagcga 960
gaccccacta tgggctcccc agggacaagg ggccgggcct gcaacaagac cagccgcctg 1020
ttggatggct gtggcagcct gtgctgtggc cgtgggcaca acgtgctccg gcagacacga 1080
gttgagcgct gccattgccg cttccactgg tgctgctatg tgctgtgtga tgagtgcaag 1140
gttacagagt gggtgaatgt gtgtaag 1167
Claims (7)
1.一种促毛发生长的组合物,其特征在于:所述组合物的活性成分包括Laminin-511修饰的间充质干细胞培养上清液、FGF10修饰的间充质干细胞培养上清液以及Wnt-10B修饰的间充质干细胞培养上清液,体积比为1:1:1。
2.根据权利要求1所述的一种促毛发生长的组合物,其特征在于:所述Laminin-511修饰的间充质干细胞培养上清液中,Laminin-511蛋白的浓度为608ng/mL;所述FGF10修饰的间充质干细胞培养上清液中FGF10蛋白的浓度为771 ng/mL;所述Wnt-10B修饰的间充质干细胞培养上清液中Wnt-10B蛋白的浓度为666 ng/mL。
3.根据权利要求1所述的一种促毛发生长的组合物,其特征在于:所述Laminin-511修饰的间充质干细胞,采用Laminin-511质粒转染间充质干细胞;所述Laminin-511质粒包括Laminin-5α质粒和Laminin-(β+γ)质粒;所述FGF10修饰的间充质干细胞,采用FGF10质粒转染间充质干细胞;所述Wnt-10B修饰的间充质干细胞,采用Wnt-10B质粒转染间充质干细胞。
4.根据权利要求3所述的一种促毛发生长的组合物,其特征在于:所述Laminin-5α的核酸人工序列为序列表中SEQ ID NO.2所示;所述Laminin-(β+γ),包括1β核酸人工序列、1γ核酸人工序列;所述1β核酸人工序列为序列表中SEQ ID NO.3所示,所述1γ核酸人工序列为序列表中SEQ ID NO.4所示;所述FGF10 的核酸人工序列为序列表中SEQ ID NO.5所示,所述Wnt-10B核酸人工序列为SEQ ID NO.6所示。
5.根据权利要求1所述的一种促毛发生长的组合物,其特征在于:所述组合物中还包括50 μg/mL的乳铁转运蛋白、50 μg/mL的生物素、8 μg/mL的维生素E、6 μg/mL的维生素B5、1μg/mL的地高辛。
6.一种促毛发生长的组合物的制备方法,其特征在于:将权利要求5所述的活性成分按照体积比为1:1:1混合,加入乳铁转运蛋白、生物素、维生素E、维生素B5、地高辛,混合均匀得到组合物。
7.一种促毛发生长的组合物的应用,其特征在于:权利要求5所述的组合物用于促进毛囊和头发生长,抑制脱发。
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