人外泌汗腺来源iPS细胞向汗腺细胞分化机制的研究

When patients get a large area deep burn, the complex structure of sweat glands cannot be regenerated during self repair. The scar tissue formed replace the original skin, and make the skin lose normal function that discharge sweat, seriously affecting the quality of life of patients. At present, the existing methods of stem cells differentiated into sweat gland cells were confronted with some problems, such as the low differentiation efficiency, and the mechanism was not clear. This cannot meet the demand for a large number of functional sweat glands during clinical treatment. Our previous work established human eccrine sweat gland cell culture methods in vitro. We established method to construct iPS cells. We also established a method differentiated human amniotic fluid stem cells into sweat gland-like cells. On the basis of preliminary work, this project aims to construct human sweat gland cell-derived iPS cells. According to the characteristics of iPS cells with high proliferation ability and cells memories of sweat gland cells, we carry out the study of SG-iPS cells differentiated into sweat gland cells. During the differentiation, explore the key molecular mechanism, to establish a efficiently methods of stem cells differentiated into sweat gland cells, improve the existing differentiation efficiency. This research may make the functional sweat gland reconstruction to be possible and may improve the clinical efficacy in treatment of patients with large area burn, and have research innovation and application potential.

当全层皮肤大面积深度烧伤时，自体修复时无法重建汗腺的复杂结构，产生疤痕组织取代原有的皮肤，使皮肤丧失正常的排汗功能，严重影响病人生活质量。目前现有的干细胞向汗腺细胞分化的方法存在分化效率低，机制不明确等问题，无法满足临床救治对功能性汗腺大量的需求。我们前期的工作建立了人外泌汗腺细胞体外分离培养方法；建立了iPS细胞构建的方法；建立了人羊水干细胞向汗腺分化的方法。在此基础上，本项目旨在通过构建人外泌汗腺细胞来源的iPS细胞，根据iPS细胞具有高增殖能力和具有来源细胞记忆性的特性，开展汗腺细胞来源的iPS细胞分化为汗腺样细胞的研究，并在分化的过程中，探讨分化的关键分子机制，建立高效的干细胞向汗腺细胞分化方法，提高现有的分化效率，使汗腺的功能性重建成为可能，提高大面积烧伤病人救治后的临床疗效，具有研究的创新性和潜在的应用价值。

皮肤组织工程中功能性汗腺的重建工作是当今医学研究的热点和难点问题，汗腺细胞体外培养增殖传代难的问题使得汗腺的重建缺乏种子细胞，干细胞向汗腺细胞分化的研究取得了一定的进展，但是在培养体系，分化效率和分化机制上还存在分歧。鉴此，结合我们的前期实验结果，本项目成功从手术切除的婴幼儿皮肤样本中分离提取出大量的汗腺组织，满足实验研究的需要，同时证明体外分离培养的汗腺细胞具有一定的增殖能力，对体外培养的汗腺细胞生物学特性进行鉴定，发现其表达汗腺特异性指标CEA，表达上皮细胞标记K14、K8和K18,具有上皮细胞的特性，以上研究结果为进一步研究汗腺细胞奠定基础；本项目利用iPS技术成功构建了人汗腺细胞来源iPS细胞，并对其生物学特性进行了检测分析，与人胚胎干细胞相似，同时定向诱导人汗腺细胞来源iPS细胞分化为汗腺样细胞，光学显微镜、Realtime-PCR、免疫荧光染色、透射电镜、3D组织培养和动物实验结果显示，人汗腺细胞来源iPS细胞能分化为汗腺样细胞，并且具有形成汗腺样结构的能力；本项目还发现人汗腺细胞来源iPS细胞分化为汗腺样细胞的过程中，HGF和BMP4信号通路参与了汗腺的分化：HGF与细胞膜上的受体c-Met结合，促进细胞基质中β-catenin表达上升，β-catenin进入细胞核内与Lef1作用，激活EDA/EDAR信号通路，从而启动汗腺分化；BMP4与细胞膜上的受体BMPR1和BMPR2结合，激活MAPK通路中的其中一条ERK通路，随后ERK与细胞核中的Lef1作用激活EDA/EDAR信号通路，从而启动汗腺分化。综上，本项目所获研究结果将为干细胞汗腺细胞分化机制的研究提供新的理论依据，也为汗腺的功能性重建开拓新的途径，具有潜在的应用价值。