毛囊干细胞及其微环境是毛囊发育和再生的基础。研究发现，病理性脱发毛囊干细胞的生存微环境被破坏是重要原因，目前缺少有效的治疗方法，而恢复或增强毛囊干细胞活化的信号有可能成为一种有效的毛囊再生方法。本研究探索将He脉冲低温等离子体（NTP）作为一种脱发治疗的新方案，并深入研究了其对毛囊干细胞和毛囊再生微环境的影响及其作用机制。本研究首先跨学科共同研发了适用于本课题研究的新型脉冲低温等离子体射流装置，用于小鼠皮肤研究，结合毛囊干细胞基因工程鼠模型，探索了NTP对皮肤损伤修复和毛囊激活的影响，研究取得以下结果：（1）He脉冲低温等离子体在不造成皮肤永久性损伤的条件下，能够稳定释放高浓度的N、N2+、He、OH和O等活性粒子；（2）运用伤口切口模型和伤口夹板模型，证明了NTP能够显著促进皮肤损伤的修复，且伤口愈合速率与NTP处理的时间和介入时伤口愈合所处的阶段有关；（3）结合Lgr5-GFP-Cre-ER和K14-H2BGFP转基因工具鼠，证明了NTP通过激活毛囊干细胞，诱导毛囊从休止期向生长期转变，进而促进毛囊再生，且效果与NTP处理的剂量呈正相关性；（4）研究揭示了NTP促进毛囊再生的生物学机制，证明NTP引起表皮细胞一过性损伤是其介导毛囊再生的基础，处理局部损伤的细胞释放激活毛囊再生的信号分子如HMGB1，促进毛囊干细胞分裂增殖，引起毛囊再生；HMGB1抑制剂显著减弱了NTP诱导的毛囊激活；（5）转录组学分析、蛋白芯片检测分析及进一步的实验证据表明，NTP可能通过介导炎症反应和血小板激活来参与毛囊再生；炎症抑制剂和血小板抑制剂显著减弱NTP诱导的毛囊激活。综上所述，我们发现NTP可以激活毛囊干细胞，诱导毛囊再生，并证明NTP介导表皮细胞释放HMGB1和诱导局部免疫细胞浸润是毛囊激活的重要机制。本研究为进一步研究基于NTP的毛囊和皮肤再生治疗方法奠定了基础。本研究利用自主研制的脉冲等离子体设备产生低温等离子体(NTP)，并将其作为皮肤再生和脱发治疗的新方案，得出以下结论：（1）通过对其光谱诊断和分析发现该NTP中有大量的活性粒子，例如含N、O、He、OH基团等活性粒子，利用其对两种伤口模型和毛囊处于顽固休止期的小鼠进行处理，发现NTP可以加速伤口的愈合，激活毛囊干细胞，显著促进毛囊周期休止期到生长期转变和毛发再生。（2）NTP促进毛发再生与输入电压、处理时间、气体流速、电极管型、轴向距离等参数密切相关，在14V、流速4L/min、连续处理三天每次3min的条件下具有较好的治疗效果。（3）
Hair follicle stem cells （HFSCs） and their microenvironment play an important role in the development and regeneration of hair follicles. Studies have found that the pathological alopecia are caused by the destruction of microenvironment surrounding HFSCs in most cases. Currently, there is a lack of effective treatment for hair follicle regenerations. Restoring or enhancing the signal of HFSCs activation might be a effective choice. In this study, He pulsed non-thermal plasma （NTP） was proposed as a new treatment for hair loss, and the effect and mechanism of NTP on HFSCs activation and hair follicle regeneration were explored and deeply studied.A new type of pulsed low temperature plasma device was firstly developed for mouse skin research. The effects of NTP on skin injury repair and activation of hair follicle cycle was accessed in HFSCs transgenic mouse model. It was found that:（1） He pulsed low temperature plasma stably released high concentration active particles such as N, N2＋, He, OH and O without causing permanent skin damage;（2） Using wound incision model and wound splint model, we proved that NTP could significantly promote the repair of skin injury, and the wound healing rate was related to the dose of NTP treatment and the stage of wound healing;（3） Using Lgr5-GFP-Cre-ER and K14-H2BGFP transgenic mice, we proved that NTP could induce telogen-to-anagen transition by activating hair follicle stem cells, thereby promoting hair follicle regeneration, and the effect was positively correlated with the dose of NTP treatment; （4） Further study proved that transient damage of epidermal cells induced by NTP was the basis of NTP-induced hair regeneration. NTP caused micro-damage of local epidermal cells, released signaling molecules such as HMGB1 which activated hair follicle regeneration, that further promoted the proliferation of HFSCs, resulting in hair follicle regeneration. The antagonist of HMGB1 significantly attenuated NTP-induced follicular activation;（5） Through transcriptome analysis, protein chip detection and further experimental evidence, we proved that NTP participated in follicular regeneration by mediating inflammatory response and platelet activation. Platelet inhibitors significantly reduced NTP-induced follicular activation. Inhibition of inflammatory response and platelet activation significantly reduced NTP-induced follicular activation.In summary, we found that NTP could activate hair follicle stem cells and induce hair follicle regeneration. Further study proved that NTP promoted hair follicle activation by inducing epidermal releasing HMGB1 and local immune cell infiltration. This study laid the foundation for further study of NTP-based hair follicle and skin regeneration therapy.
