海藻酸钠水凝胶三维培养在维持毛乳头细胞诱导毛囊再生能力中的作用及机制研究

Freshly isolated human dermal papilla (DP) has been shown to be able to induce de novo hair growth. However, it quickly loses its hair-inducing properties upon in vitro expansion, which greatly limits its clinical application for hair follicle reconstruction. In the preliminary study, we established a three-dimensional (3D) culture system with sodium alginate hydrogel mainly composed of alginate, a kind of natural polysaccharide extracted from brown seaweed, and found that human DP cells cultured in this system would aggregate into microspheres and the hair-inducing ability of DP cells was at least significantly recovered. In this study, to obtain the optimal conditions for DP culture, we will continue to adjust the preparation of sodium alginate hydrogels. The expression of hair-inductive markers will be analyzed in DP cells at different passages cultured in adjusted sodium alginate microspheres by quantitative RT-PCR, immunostaining and immunoblotting. Then, these DP cells will be co-implanted with newborn mouse epidermal cells or human low-passage keratinocytes into nude mice to evaluate their hair-inducing ability. In addition, to seek the key signals involved in the restoration of hair-inducing properties of DP cells cultured in the optimal 3D culture system, we intend to execute RNA-seq analysis of DP cells at monolayered culture (2D) and microspheres (3D) at various time points. Through gain- and loss-of function approaches in vitro, together with hair follicle reconstitution in vivo, we will explore the roles of the key genes from RNA-seq analysis in maintaining or restoring the hair-inducing properties of DP cells at 2D and 3D culture. Finally, by generating conditional transgenic mice, we will explore the roles of several indicated key genes in the DP in hair regeneration. Our ultimate goal is to establish an ideal culture system for DP cells in vitro and figure out the key signals during maintenance and restoration of inductive properties, which will provide new cues for hair loss treatment.

新鲜分离的人毛乳头（DP）能诱导毛囊再生，但在体外逐渐丧失这种功能，极大限制了临床上大量重建毛囊治疗患者。海藻盐是一种天然多糖，我们将其制备成海藻酸钠水凝胶并对其进行了初步改造，发现人DP细胞在其中3D培养后会聚集成球，毛囊诱导能力显著恢复。本研究中，我们首先将继续调整海藻酸钠水凝胶的性状，将不同代数人DP细胞培养在不同的海藻酸钠微球中，检测毛囊诱导相关分子的表达，同时与小鼠或人的角质形成细胞混合注入裸鼠皮下观察毛囊再生情况，以得到最佳的水凝胶制备条件；再在不同时间点取上述培养DP细胞的RNA行RNA-seq检测以寻找恢复诱导能力的重要分子，然后在不同培养方式培养的DP细胞中进行调节并在裸鼠体内验证其毛囊诱导能力；最后制备DP特异性敲除/敲入鼠以验证其功能。本研究的最终目的在于建立理想的DP体外培养条件并寻找维持和恢复其毛囊再生诱导能力的关键因子，为临床上脱发性疾病的治疗提供新思路。

目前药物治疗和自体毛囊移植这两种治疗脱发的方法不能促进新的毛囊的形成。真皮毛乳头细胞（DPCs）在毛囊形态发生和调节毛发循环中起着至关重要的作用，但是体外传代培养会快速丢失其诱导毛发生成的能力。所以，如何在体外获得大量具有诱导毛发再生能力的DPCs已经成为目前毛囊再生研究的热点和难点。.本研究的主要目的是探究海藻酸钠的三维培养环境是否能够诱导真皮成纤维细胞向毛乳头样细胞的转化及可能的作用机制。为促进成纤维细胞的聚集，我们制备包裹真皮成纤维细胞的海藻酸-多聚赖氨酸-海藻酸（APA）微球，检测人真皮成纤维细胞的增殖，人毛乳头特异性基因的表达和人毛乳头标记基因的蛋白表达水平。发现人真皮成纤维细胞的DPC标记基因在转录和蛋白水平都随着APA微球培养时间的增加而增强。.为探究人毛乳头样细胞是否具有诱导毛发再生的能力，诱导产生的毛乳头样细胞和新生鼠表皮细胞共同通过皮下注射的方法注射到裸鼠背部来观察新生毛发的再生情况。发现4周后裸鼠背部能够产生类似于阳性对照的黑色团状结构和毛囊样结构，并且人线粒体标记进一步确定新生毛囊结构至少部分来自于人毛乳头样细胞，同时毛乳头标记性基因的染色也进一步说明产生的毛囊样结构具有毛发的功能。随后对人毛乳头细胞、人真皮成纤维细胞及人毛乳头样细胞进行了转录组水平的测序分析，结果表明人真皮成纤维细胞向人毛乳头样细胞转化过程中 Wnt 信号通路激活，并且APA三维培养环境下分别加入Wnt信号的抑制剂或激活剂后DPC特异性基因也随之分别发生下调或上调，揭示了Wnt信号通路在人真皮成纤维细胞向人毛乳头样细胞转化过程中起着重要作用。此外，我们发现APA微球处理的鼠真皮成纤维细胞同样能够转化为鼠毛乳头样细胞。并且与鼠新生鼠表皮细胞构建的毛囊再生模型后具有诱导毛囊样结构的形成和毛发色素沉着的能力，证明了APA微球诱导DF-DPC转化的普适性。.综上，本研究证明了APA微球处理能够分别诱导人/鼠成纤维细胞转化为人/鼠毛乳头样细胞，并且人/鼠毛乳头样细胞聚集和毛乳头功能活性的获得能够诱导毛囊再生，在此过程中Wnt信号通路起着重要的调控作用。通过APA微球处理得到的毛乳头样细胞能够为毛囊再生提供一种新颖、充分而且更安全的毛乳头细胞的来源。