CN111135194A - 促进毛发再生的外用药物组合物及制备方法 - Google Patents
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- CN111135194A CN111135194A CN202010168237.6A CN202010168237A CN111135194A CN 111135194 A CN111135194 A CN 111135194A CN 202010168237 A CN202010168237 A CN 202010168237A CN 111135194 A CN111135194 A CN 111135194A
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Abstract
本发明的目的在于提供一种促进毛发再生的外用药物组合物及其制备方法，其技术方案在于，包括间充质干细胞无血清培养基上清，还包括与该间充质干细胞无血清培养基上清联用的维甲酸。本发明所述的外用药物组合物主要适用于脂溢性脱发等脱发症状，通过干细胞无血清培养基上清与维甲酸联用，能够有效激发毛囊，促进脱发者的毛发再生，具有方便、安全、低成本、效果显著、治疗过程简单、病人接受度高的优势。
Description
技术领域
本发明涉及蛋白药物领域，特别涉及一种包含间充质干细胞无血清培养上清联合维甲酸的外用药物组合物及其制备方法。
背景技术
脱发是皮肤科的一种常见疾病，分为非瘢痕性脱发和瘢痕性脱发。雄激素性脱发、斑秃、拔毛癖等属于前者，毛发扁平苔藓、毛囊炎性脱发、盘状红斑狼疮性脱发等属于后者。雄激素性脱发最为常见，表现为头发密度进行性减少，为雄激素依赖的常染色体显性遗传性多变性疾病。
目前针对脱发患者有几种治疗选择，如使用口服或者外用药物，或者进行手术治疗。然而，这些治疗方式具有局限性，药物治疗只能短暂的缓解症状，当病人停止用药后头发又会开始脱落。比如口服非那雄胺有性副作用并且会至畸，停止用药后，头发会加速脱落。采用自体的单个毛囊和毛囊单位移植是一种可靠的手术治疗方法，但是毛囊供体的数量是有限的，并且毛囊移植存活率低而费用高，并且会给病人带来极大的痛苦。因此，脱发急需一些新的治疗手段。
发明内容
本发明的目的在于提供一种促进毛发再生的外用药物组合物及其制备方法，能够有效针对应脱发，有效促进脱发者毛发再生。
本发明采用的具体方案为：一种促进毛发再生的外用药物组合物，包括间充质干细胞无血清培养基上清，其技术方案在于，还包括与该间充质干细胞无血清培养基上清联用的维甲酸。
所述的维甲酸的质量分数0.024%~0.026%。
一种如上述外用药物组合物的制备方法，其技术方案在于，包括以下步骤：将干细胞无血清培养基上清分装后置于4℃保存，与质量分数0.024%~0.026%的维甲酸进行混合，得到最终药物组合物。
所述的间充质干细胞无血清培养基上清的制备过程如下：
S1.冲洗脐带，去除脐带表面残留的血液，剪成5×5×5毫米以下的块状，均匀涂布于培养皿上，再缓慢加入α-MEM完全培养基进行培养，确保脐带组织可以更加均匀，得到待培养样本；
S2.将S1步骤的待培养样本置于温度为37℃且体积分数为5％的二氧化碳饱和湿度培养箱内，直至干细胞长出约占满细胞培养皿75~85 %面积后，使用0.25 %胰酶消化传代；
S3.待S2步骤的胰酶消化传代传至第三代后，培养48 h后采用低速离心法获取上清，最终得到间充质干细胞无血清培养基上清。
S1步骤中所述的α-MEM完全培养基的培养体系为：α-MEM基础培养基中加入谷氨酰胺 1.8~2.2mM；氢化可的松 49~51μg/L；硫酸锌 0.9~1.1mg/L；丁酸钠 1.9~2.1mM；柠檬酸铁 190~210μM；维生素C 190~210μM；重组人表皮生长因子 19~21μg/L；重组人碱性成纤维细胞生长因子 19~21μg/L；RGD短肽 19~21μg/L；棕榈酰三肽49~51μg/L；棕榈酰四肽39~41μg/L；氯化锂4~6mM；L-谷胱甘肽 3~5mg/L；大豆胰酶抑制剂 90~110mg/L；β-巯基乙醇 2.4~2.6mg/L；乙醇胺 0 .1~0.3g/L；亚硒酸钠 0.00066~ 0.00068mg/L；丙酮酸钠 0.08~0.12mM；Hepes 0.008~0.012M；NaHCO 32~34mM。
有益效果：本发明所述的外用药物组合物主要适用于脂溢性脱发等脱发症状，通过干细胞无血清培养基上清与维甲酸联用，能够有效激发毛囊，促进脱发者的毛发再生。具有方便、安全、低成本、效果显著、治疗过程简单、病人接受度高的优势。
附图说明
图1为8周后头发增量对照表。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。
脱发是一种炎性细胞因子介导疾病，脱发的发病机制与Th-1和NKG2D介导的信号传导密切相关。研究显示，毛囊是具有免疫豁免的部位，当毛囊的免疫豁免崩溃时，CD4CD8T细胞和一些自然杀伤细胞聚集在毛球自身抗原周围并会导致脱发的形成。
间充质干细胞具有细胞免疫调节和抗炎的作用，科学家通过试验获取成功后，通过干细胞临床治疗，结果发现干细胞能抑制IFNG，CXCL9和CXCL10的产生和NKG2D+T细胞的渗透，可以有效预防脱发。干细胞可分泌大量生长因子，如角质细胞生长因子、血管上皮细胞生长因子、血小板生长因子和肝细胞生长因子等，这些生长因子蛋白在促进毛发再生的过程中起着重要作用。但是干细胞移植只能到拥有相关资质的医疗机构或者整形机构进行，这无疑增加了脱发的治疗成本。有研究表明在干细胞的培养过程中，也会向培养基中分泌上述的治疗因子，因此可以采用干细胞培养基上清来达到上述的治疗目的。在这一过程中同样存在一个问题，干细胞培养基中的一般添加动物血清（胎牛血清），但是胎牛血清为动物来源物质，会给干细胞培养基带来多种不利因素，如不同批次发明差异大，来源不稳定，且动物血清也有可能成为一种过敏原影响人的健康。因此本发明所述的外用药物组合物采用干细胞无血清培养基上清，此种培养基中的物质成分完全已知，可以在达到治疗目的的同时，有效避免上述问题。
维A酸（维甲酸）是体内维生素A的代谢中间产物，主要影响骨的生长和促进上皮细胞增生、分化、角质溶解等代谢作用。维A酸的药理作用主要是由RARs和RXRs受体介导的反式激活或转录抑制表现出来。反式激活包括配体结合、形成二聚物（RAR/RXR）、与DNA的相互作用、招募共激活剂和RNA延伸等5个步骤完成的。不同的维A酸具有不同的受体结合特性。维A酸类药物具有以下药理作用：①调节细胞增殖和分化，抗异常角化；②改变细胞粘附性；③诱导细胞凋亡，是抑制皮脂腺功能及抗肿瘤的重要机制；④抗炎症反应及免疫调节，特别是具有影响Toll样受体表达和调节多种亚型的T细胞，从而影响到天然免疫或获得性免疫；⑤影响细胞外基质代谢；⑥抗血管或淋巴管生成。这些药理作用成为多种皮肤病特别是皮脂腺相关疾病、炎症性疾病、角化性疾病及皮肤肿瘤的重要基础。
基于上述内容，本发明提供一种适用于脂溢性脱发等脱发症状的能够有效促进脱发者毛发再生的外用药物组合物，具有稳定、高效且价格低廉的优点。
具体实施例I：本发明所述的外用药物组合物包括间充质干细胞无血清培养基上清与维甲酸构成的组合物。
一）间充质干细胞无血清培养基的制备：
在α-MEM基础培养基中加入谷氨酰胺 1.8mM；氢化可的松 49μg/L；硫酸锌 0.9mg/L；丁酸钠 1.9mM；柠檬酸铁 190μM；维生素C 190μM；重组人表皮生长因子 19μg/L；重组人碱性成纤维细胞生长因子 19μg/L；RGD短肽 19μg/L；棕榈酰三肽49μg/L；棕榈酰四肽39μg/L；氯化锂4mM；L-谷胱甘肽 3mg/L；大豆胰酶抑制剂 90mg/L；β-巯基乙醇 2.4mg/L；乙醇胺 0.1g/L；亚硒酸钠 0.00066mg/L；丙酮酸钠 0.08mM；Hepes 0.008M；NaHCO 32mM；
或α-MEM基础培养基中加入谷氨酰胺 2.2mM；氢化可的松 51μg/L；硫酸锌 1.1mg/L；丁酸钠2.1mM；柠檬酸铁 210μM；维生素C 210μM；重组人表皮生长因子 21μg/L；重组人碱性成纤维细胞生长因子 21μg/L；RGD短肽 21μg/L；棕榈酰三肽51μg/L；棕榈酰四肽41μg/L；氯化锂6mM；L-谷胱甘肽 5mg/L；大豆胰酶抑制剂 110mg/L；β-巯基乙醇 2.6mg/L；乙醇胺0.3g/L；亚硒酸钠0.00068mg/L；丙酮酸钠 0.12mM；Hepes 0.012M；NaHCO 34mM。
或所述的α-MEM完全培养基的最优培养体系为：谷氨酰胺 2mM；氢化可的松 50μg/L；硫酸锌 1mg/L；丁酸钠 2mM；柠檬酸铁 200μM；维生素C 200μM；重组人表皮生长因子 20μg/L；重组人碱性成纤维细胞生长因子 20μg/L；RGD短肽 20μg/L；棕榈酰三肽50μg/L；棕榈酰四肽40μg/L；氯化锂 5mM；L-谷胱甘肽 4mg/L；大豆胰酶抑制剂 100mg/L；β-巯基乙醇2.5mg/L；乙醇胺 0 .2g/L；亚硒酸钠 0.00067mg/L；丙酮酸钠 0.1mM；Hepes 0.01M；NaHCO33mM。
二）间充质干细胞无血清培养基上清的获取：
脐带带回后，在超净工作台中进行操作，使用含有2倍100U/mL青霉素和链霉素的生理盐水进行冲洗脐带，去除脐带表面残留的血液，剪成5×5×5毫米以下的块状，均匀涂布于培养皿上，再缓慢加入无血清培养液进行培养，确保脐带组织可以更加均匀，同时也是使细胞培养过程中，为细胞提供良好的生长空间，使细胞的生长不受限制，另一方面也确保细胞培养的数量足够。置于温度为37℃，体积分数为5％的二氧化碳饱和湿度培养箱直至干细胞长出约占满细胞培养皿80 %左右面积后使用0.25 %胰酶消化传代，期间每隔3-4天进行培养基换液。待传至第三代后，培养48 h后采用低速离心法即2000r/min 离心15min，获取上清。将获得的干细胞无血清培养基上清分装后置于4℃保存。
三）间充质干细胞无血清培养基上清与维甲酸混合：
在混合时控制维甲酸的质量分数0.025%或0.024%或0.026%或0.024%~0.026%中的任意数值，得到最终的目标组合物。
本发明的原理说明：
本发明外用药物组合物通过干细胞无血清培养基上清与维甲酸联用，干细胞无血清培养基中的大量生长因子，如角质细胞生长因子、血管上皮细胞生长因子、血小板生长因子和肝细胞生长因子等。同时维甲酸能够抑制皮脂腺分泌可使皮脂腺的基底细胞成熟过程变长，皮脂腺数目减少，皮脂腺中增殖细胞的比例下降，导致皮脂合成减少。此外也可以可使中性粒细胞游走受到抑制，抑制花生四烯酸及其代谢产物的产生，抑制中性粒细胞产生白三烯及氧化物阴离子，阻碍溶酶体的释放，消除炎症。因此二者联用有效改善脱发者头皮处的毛囊微环境，使得其适宜毛发生长，能够有效激发毛囊，促进脱发者的毛发再生。
毛发再生实验：
一）分组规则：
脱发者来源及分组：脱发者为8-30岁患者20例，31-40岁患者20例，41-50岁患者20例，51岁以上患者20例，共计80例。每组20人，共计四组。
遵循每个组别中各个年龄段患者均匀分布，将患者随机分成4组，其中：
实验组为使用间充质干细胞无血清培养基上清与维甲酸混合的治疗方法；
实验组2为单一使用充质干细胞无血清培养基上清的治疗方法；
实验组3为单一使用维甲酸的治疗方法，控制维甲酸含量与实验组相同，溶剂为无菌去离子水；
阳性对照组为使用市面上常见的药物（蔓迪米诺地尔酊）。
二）给药方法
使用时，晚上将头发洗净，涂抹本发明所述的外用药物组合物3mL，按摩5分钟，第二天早上将残留液洗净，每周使用3次。此过程中应注意控制其他非药物因素，避免干扰；另，阳性对照组药膏使用方法按照说明书使用。
三）效果评估：
（1）有效性评估
显著有效：脱发停止，长出较多新发
有效：脱发症状减轻并伴随有新发长出
无效：脱发情况没有改善
在共计四周的实验过程中，每7天对患者治疗脱发效果进行评估。
（2）发密度评估
发密度：一平方厘米头皮有多少个毛囊单位，可以使用专业仪器检测。
检测部位为出现脱发状态的头皮部分，测试条件为室温（20-25℃），湿度为46-50%。将脱发部位直径为1cm的圆状区域内，头发长度处理为2mm或者更小的长度，标记出面积为1cm2的测试部位，为了保证测试条件相同，每次测量时，待测者需在测试房间等候休息30min，且每次测量部位相同。测试部位的发密度数量变化作为其评估标准。
效果验证：
（1）有效性评价
在为期四周的实验过程中，每两周对患者脱发处进行观察并且询问使用情况，使用本发明发明的脱发者均无瘙痒，红肿等不良反应，在实验组20例患者中的19人脱发情况得到显著改善，其余一人部分改善，远远高于实验组2，实验组3，和阳性对照的数据。实验组2中有一例患者情况没有改善，实验组3中有一例患者情况没有改善，阳性对照组中有2例患者情况没有改善。具体情况如下表I所示：
表I：
（2）发密度评估
实验共计进行8周，每隔两周对脱发者的脱发部位相同区域进行评估，其中实验组的人员发密度增加远远超过其他组别，在8周后增加量为16根/cm2，实验组2，实验组3，阳性对照组分别为10、8、 9根/cm2，具体情况如图1所示。
根据图1和表I可知，将间充质干细胞无血清培养基上清与维甲酸联用后，能够有效改善患者脱发情况，其效果远高于二者单独使用，并且适用于各个年龄段的人群使用，使用一段时间后，脱发患者的脱发症状得到显著改善，并且新生头发量较多，可以作为一种治疗脱发和促进头发生长的新的方法。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易变化或替换，都属于本发明的保护范围之内。因此本发明的保护范围所述以权利要求的保护范围为准。
Claims (6)
1.一种促进毛发再生的外用药物组合物，包括间充质干细胞无血清培养基上清，其特征在于，还包括与该间充质干细胞无血清培养基上清联用的维甲酸。
2.根据权利要求1所述的促进毛发再生的外用药物组合物，其特征在于，所述的维甲酸的质量分数0.024%~0.026%。
3.一种如权利要求1所述促进毛发再生的外用药物组合物的制备方法，其特征在于，包括以下步骤：将干细胞无血清培养基上清分装后置于4℃保存，与质量分数0.024%~0.026%的维甲酸进行混合，得到最终药物组合物。
4.根据权利要求3所述的制备方法，其特征在于：所述的间充质干细胞无血清培养基上清的制备过程如下：
S1.冲洗脐带，去除脐带表面残留的血液，剪成5×5×5毫米以下的块状，均匀涂布于培养皿上，再缓慢加入α-MEM完全培养基进行培养，确保脐带组织可以更加均匀，得到待培养样本；
S2.将S1步骤的待培养样本置于温度为37℃且体积分数为5％的二氧化碳饱和湿度培养箱内，直至干细胞长出约占满细胞培养皿75~85 %面积后，使用0.25 %胰酶消化传代；
S3.待S2步骤的胰酶消化传代传至第三代后，培养48 h后采用低速离心法获取上清，最终得到间充质干细胞无血清培养基上清。
5.根据权利要求4所述的制备方法，其特征在于：S1步骤中所述的α-MEM完全培养基的培养体系为：α-MEM基础培养基中加入谷氨酰胺 1.8~2.2mM；氢化可的松 49~51μg/L；硫酸锌 0.9~1.1mg/L；丁酸钠 1.9~2.1mM；柠檬酸铁 190~210μM；维生素C 190~210μM；重组人表皮生长因子 19~21μg/L；重组人碱性成纤维细胞生长因子 19~21μg/L；RGD短肽 19~21μg/L；棕榈酰三肽49~51μg/L；棕榈酰四肽39~41μg/L；氯化锂4~6mM；L-谷胱甘肽 3~5mg/L；大豆胰酶抑制剂 90~110mg/L；β-巯基乙醇 2.4~2.6mg/L；乙醇胺 0 .1~0.3g/L；亚硒酸钠0.00066~ 0.00068mg/L；丙酮酸钠 0.08~0.12mM；Hepes 0.008~0.012M；NaHCO 32~34mM。
6.根据权利要求4或5所述的制备方法，其特征在于：所述的α-MEM完全培养基的最优培养体系为：谷氨酰胺 2mM；氢化可的松 50μg/L；硫酸锌 1mg/L；丁酸钠 2mM；柠檬酸铁 200μM；维生素C 200μM；重组人表皮生长因子 20μg/L；重组人碱性成纤维细胞生长因子 20μg/L；RGD短肽 20μg/L；棕榈酰三肽50μg/L；棕榈酰四肽40μg/L；氯化锂 5mM；L-谷胱甘肽 4mg/L；大豆胰酶抑制剂 100mg/L；β-巯基乙醇 2.5mg/L；乙醇胺 0 .2g/L；亚硒酸钠0.00067mg/L；丙酮酸钠 0.1mM；Hepes 0.01M；NaHCO33mM。
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| CN112263673A (zh) * | 2020-11-03 | 2021-01-26 | 华科同济干细胞基因工程有限公司 | 一种促进毛发再生的外用药物及其制备方法 |
| CN116688100A (zh) * | 2023-07-12 | 2023-09-05 | 北京益华生物科技有限公司 | 一种促进毛囊再生的组合物及制备方法和应用 |
| WO2024107102A1 (en) * | 2022-11-17 | 2024-05-23 | Cellresearch Corporation Pte. Ltd. | Use of conditioned medium derived from cultivation of mesenchymal stem cells of the umbilical cord for inducing, stimulating and promoting hair growth and regeneration |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112263673A (zh) * | 2020-11-03 | 2021-01-26 | 华科同济干细胞基因工程有限公司 | 一种促进毛发再生的外用药物及其制备方法 |
| WO2024107102A1 (en) * | 2022-11-17 | 2024-05-23 | Cellresearch Corporation Pte. Ltd. | Use of conditioned medium derived from cultivation of mesenchymal stem cells of the umbilical cord for inducing, stimulating and promoting hair growth and regeneration |
| WO2024107122A1 (en) * | 2022-11-17 | 2024-05-23 | Cellresearch Corporation Pte. Ltd. | Use of conditioned medium derived from cultivation of mesenchymal stem cells of the umbilical cord for inducing, stimulating and promoting hair growth and regeneration |
| CN116688100A (zh) * | 2023-07-12 | 2023-09-05 | 北京益华生物科技有限公司 | 一种促进毛囊再生的组合物及制备方法和应用 |
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