基于间充质干细胞囊泡促进毛囊再生的关键技术研究

Hair regeneration is a worldwide medical problem, and the desire to regenerate hair is at the root of a multi-billion dollar industry worldwide. However, there are currently few truly effective treatments due to their limitations of inefficiencies and potential adverse side effects, which not only affect patients' mental health, but also endanger certain inherent functions of the skin. In the preliminary study of this project, it was found that proinflammatory cytokines TNF-α and IL-1α are important microenvironmental factors in hair follicle, which interact with surrounding cells to activate stem cells in hair follicle and promote hair follicle regeneration through paracrine mechanism. At the same time, preliminary study also showed that three-dimensional culture (3D) significantly increased the paracrine capacity and the secretion of extracellular vesicles from mesenchymal stem cells (MSC-EV). Accordingly, this project is intended to introduce the two pretreatment factors into 3D MSC culture system in order to cause the greater change of secretome from MSC, which aim to increase the secretion of MSC-EV and significantly change the contents in EV. MSC-EV from the optimized culture condition contained a variety of hair follicle regeneration related factors, which enhance the efficacy of MSC-EV for the hair loss treatment. In this project, in vitro and in vivo experiments will be conducted to verify the effect and the molecular mechanism of extracellular vesicles derived from TNF-α/IL-1α priming Mesenchymal Stem Cell Spheroids on hair follicle regeneration. In this project, the MSC-EV derived from the optimized culture condition will represent mixed factors with specific efficacy to enhance hair follicle regeneration, which is expected to be a very significant new treatment strategy for hair loss in hair regeneration medicine, with great biological significance and clinical application value.

头发再生是世界性的医学难题，重新生长头发的愿望是全球数十亿美元产业的根源，然而受低效和潜在不良副作用的限制，目前几乎没有真正有效的治疗方法，不仅影响患者的心理健康，而且还危及皮肤的某些固有功能。本项目前期研究显示3D培养显著增加间充质干细胞旁分泌能力及囊泡（MSC-EV）分泌量；同时发现，TNF-α和IL-1α是构成毛囊中的重要微环境因子，其与周围细胞相互作用，通过旁分泌方式激活毛囊中干细胞，促进毛囊再生。据此，本项目欲在MSC的3D培养体系中引进这两种预处理因子，旨在增加囊泡产量并显著改变囊泡内容物特性，增强其毛囊再生功效；然后再通过体内外实验证实该培养方案来源的囊泡对毛囊再生的影响，并揭示其在毛囊再生中的分子机制。本项目中优化培养方案来源的MSC-EV将代表一种具有特定增强毛囊再生功效的混合因子制剂，有望成为头发再生医学中非常有意义的脱发治疗新策略，具有重大生物学意义和临床应用价值。

毛发再生是世界性的医学难题，重新生长头发的愿望是全球数十亿美元产业的根源，然而受低效和潜在不良副作用的限制，目前几乎没有真正有效的治疗方法，不仅影响患者的心理健康，而且还危及皮肤的某些固有功能。本项目在MSC的3D培养体系中引进TNF-α和IL-1α这两种毛囊中的重要微环境因子，建立优化了一种新型的 3D 培养技术，结果显示，与传统的 2D 培养相比，3D 培养与预处理因子 TNF-α和 IL-1α引入相结合的培养方式显著增加了胞外囊泡的分泌量，其产率是2D培养条件下的 7 倍左右。本研究分离提取来自此优化培养方案来源的MSC-EV，通过体外实验检测其对毛囊干细胞激活和对 DP 细胞增殖、碱性磷酸酶 AP 活性的影响。结果发现TNF-α/IL-1α预处理的 3D MSC-EV 促进毛囊干细胞集落形成，促进 DP 细胞增殖存活及明显增加其AP 的活性。本研究进一步通过体内动物实验证实该培养方案来源的囊泡明显促进了毛囊再生，显著增加了毛发的数量。我们进一步对其机制进行研究，发现优化培养条件来源的MSC-EV内容物中含有更多的生长因子以及毛发诱导相关蛋白，而PCR分析结果显示优化培养条件来源的MSC-EV显著增加了毛囊诱导相关基因的表达。此外，本项目进一步找出对毛囊从头再生至关重要的关键信号通路，PI3K-Akt信号通路，并开发出一种能够促进毛囊再生及完全恢复受损皮肤结构和功能的工程皮肤替代品胶原蛋白-糖胺聚糖凝胶。这些工作为含毛囊再生的皮肤完整修复治疗新策略提供宝贵的线索，在头发再生医学及皮肤完整再生领域具有重大生物学意义和应用价值。而本项目中优化培养方案来源的具有特定增强毛囊再生功效的MSC-EV代表一种混合因子制剂，有望在临床上被用作强有力的毛发生长初期诱导剂，为脱发治疗提供一种安全高效的无细胞MSC-EV囊泡疗法，将具有重大生物学意义和临床应用价值。