Wnt/TGF-β在KGF诱导hUC-MSCs分化为汗腺样细胞SGCs过程中的调控机制及其促分化效能

The original structure and function of human skin and cutaneous appendages (like sweat glands) could be seriously destroyed by severely thermal injuries. After then, survivors might lose their functions of sweat perspiration and temperature modulation as well as their own regenerative ability. So it is a pivotal issue for us to investigate a promising way to repair and regenerate survivors’ primitive skin structure and function of injured sweat glands(SGs) and skin through differentiation and proliferation of exogenous stem cells. Based on our previous research, it was demonstrated that human umbilical cord derived mesenchymal stem cells (hUC-MSCs) could be trans-differentiated into sweat gland-like cells(SGCs) by culturing in mixing-induction medium consisted of 20% conditioned medium plus 80% basic sweat gland medium, as well as KGF-induction medium contained with recombinant human keratinocyte growth factors (rhKGF) and basic sweat gland medium. Unfortunately, it has not been clarified that the detailed roles and molecule mechanism related to KGF in differentiation of SGCs from hUC-MSCs. As a classical signal pathway during skin development, EDA/EDAR could control morphogenesis of SGs through activation and crosstalk of extracellular Wnt/TGF-β and intracellular Smads signal pathways, which could manipulate cell fate and proliferation potency. In addition, it was shown that KGF could also possess the potential to regulate Wnt/TGF-β signal pathways through activation of modulator gene (c-Myc), which might be involved in development and mature process of SGs. However, But it has not been investigated that potential mechanism related to KGF could play some important roles during differentiation of SGCs from hUC-MSCs or other sources of stem cells. In this study, dose-dependent efficiency and specificity of KGF would be demonstrated by examination of different concentration of rhKGF and its isomers (FGF-10, KGF-2) during differentiation of SGCs, as well as expression of related modulator proteins of extracellular Wnt/TGF-β and their intracellular Smads signal pathways. In addition, over- expression of promoter (c-Myc) would be applied to activate Wnt/TGF-β to evaluate the induction efficiency and molecule mechanism of SGCs from hUC-MSCs, compared with use of Wnt inhibitor(DKK4) and/or BMP inhibitor (Noggin) , which acted as downstream of TGF-β and were essential for maturation of ducts of SGs. Finally, higher differentiation-efficiency of SGCs would be achieved by proper concentration of rhKGF combined with co-regulation of Wnt/TGF-β signal pathway, which would be documented as a detailed crosstalk mechanism of differentiation of SGCs from hUC-MSCs. In a word, It would serve as detailed modulation mechanism related to KGF, useful induction system with higher efficiency, better solution for repair and regeneration of severely injured SGs by differentiation of SGCs from hUC-MSCs and other kinds of stem cells in the future.

严重烧伤破坏人体皮肤及汗腺等附属器功能，使其丧失排汗及自我修复潜能。此时，皮肤汗腺的重建需借助外源性干细胞移植完成，其成为皮肤再生研究的新方向。EDA/EDAR在皮肤发育时通过调控细胞增殖与分化蛋白Wnt/TGF-β、胞内Smads表达促进汗腺成熟；而KGF也可通过c-Myc调控Wnt/TGF-β、Smads表达完成汗腺发育。经研究发现，含rhKGF或热休克汗腺上清液培养基均可诱导hUC-MSCs分化为汗腺样细胞SGCs；但其调控机制不详。本研究将采用rhKGF 浓度梯度及同分异构体FGF10（KGF-2）探索其在SGCs分化过程中的特异性及Wnt/TGF-β、Smads分子调控机制；并通过激动剂c-Myc、抑制剂DKK4和/或Noggin协同调节Wnt/TGF-β通路用以提高SGCs分化效能。最终阐明Wnt/TGF-β在KGF诱导hUC-MSCs分化为SGCs的分子机制、提高分化效能。

严重烧伤会损伤皮肤组织结构与功能，引起患者创面愈合质量下降，影响患者生存质量。其中，汗腺组织的损伤会引起患者患者排汗功能障碍、调节功能丧失。因此，促进损伤皮肤组织汗腺结构重建与功能恢复成为皮肤组孩子修复与再生研究的重要方向之一。既往采用生长因子等方式可以促进创面愈合，采用骨髓间充质干细胞以及组织工程皮肤移植的方式可以再一定程度上修复皮肤结构与功能。本研究中采用组织来源丰富的脐带间充质干细胞hUC-MSCs进行汗腺样细胞（SGCs)诱导分化，并且对汗腺诱导分化的分子机制以及诱导分化培养基进行标准化制作。研究结果表明：1）hUC-MSCs能够在成品化的汗腺分化诱导培养基中分化成为SGCs，并且在损伤汗腺的移植修复过程中具有明显的组织修复效果；2）本研究中同样证实汗腺发育基因角化细胞生长因子KGF联合汗腺培养基能够达到制备新型汗腺分化诱导培养基的目的，并且比前期研究中利用热休克汗腺上清液-汗腺分化诱导培养基的诱导活性更加稳定；3）hUC-MSCs经过不同汗腺分化诱导培养基分化诱导过程中获取的SGCs的效率存在一定的差异，其中成品化的KGF汗腺分化诱导培养基对hUC-MSCs诱导分化为SGCs的效率以及细胞的增殖活性具有较好的效果。.此外，在本项目研究团队中成员中对严重烧伤患者的救治经验经过总结后成为严重烧伤救治的专家共识，具有一定的推广价值。另外一部分研究着眼于hUC-MSCs对于其他组织（肺脏）损伤修复的研究，并且取得了一定的研究进展。.总之，本研究团队中成员紧密围绕项目研究的主要内容开展hUC-MSCs与SGs的损伤修复基础研究与临床治疗方面的探索应用。通过前期研究结果，发现本研究的结果具有一定的推广应用价值。本项目研究团队中的其他研究成果同样围绕组织修复的方向进行探索性研究，以期将本研究的成果不断拓展入相关研究领域，促进组织修复与再生医学的发展。