基于微环境诱导人脐带间充质干细胞分化并参与早期修复损伤汗腺的相关机制研究

Sweat glands could easily be destroyed by extensive and severe traumas(such as burns),which result in loss of body temperature regulation and homeostasis maintenance. Meanwhile, skin and cutaneous appendages lost its own regeneration potential after severe injuries. If we hope to re-establish the original structure and function of skin, we have to depend on the various therapeutic ways and technologies. Nowadays, there were some methods for repairing damaged sweat glands, including differentiating autologous bone marrow derived mesenchymal stem cells(BM-MSCs) into sweat gland cells through co-culturing them with heat-shocked sweat gland cells or being transfected with sweat gland development gene(ectodermal dysplasia gene,EDA), as well as covering the damaged skin with bioengineering skin. Among them, there will be a long time for culture and differentiation of autologous BM-MSCs. If we apply the tissue engineering skin combined with sweat gland cells into repairing the damaged sweat glands, we had to solve at least two problems: immunologic rejection of allogeneous sweat glands or long culture period of autologous cells.However, there was not a positive way which could re-establish destroyed sweat glands at the early period of cutanous wound healing.. Many previous studies have demonstrated that mesenchymal stem cells derived fron human umbilical cord Wharton's jelly (hUC-MSCs) possess the great properties, such as low immune-resistance, multiple differentiation, convenient isolation and culture, which make them to be an ideal source applied in the study of sweat gland regeneration and repair. Based on our previous studies, it was found that hUC-MSCs also have the potential to differentiate into sweat gland cells under special micro-environment. In addition, Some key sweat gland development genes have been found to play important roles in the formation of sweat glands.In this study, we will dwell on the mechanism and roles of sweat gland- development genes in the differentiation of hUC-MSCs. We hope that we could find out the key gene(s) which play the povital role in inducing hUC-MSCs into sweat gland-like cells. In the latter stage, we will try to search out the proper saturation of sweat gland-development gene(s) in the sweat gland- differentiation medium, and clarify the detailed mechanism of the development gene(s) involved in the differentiation process. After then, we will culture hUC-MSCs in our novel sweat gland-induction system to obtain sweat glands, which will be used for the repair and regeneration of damaged sweat glands at the early period of cutaneus wound healing. we will evaluate the efficacy and bio-safety of the differentiation system, as well as the positive result of the transplantation of sweat gland-like hUC-MSCs. In a word, we hope that our study could make a progress for the theory of sweat gland regeneration, supply a new therapeutic way to repair destroyed sweat glands as early as possible.

重度烧创伤破坏皮肤中汗腺结构与功能，严重影响患者体温调节与排汗功能，降低其生活质量。当前利用骨髓间充质干细胞的转分化潜能成为修复损伤汗腺的一种有效途径。但是，如何在皮肤损伤早期利用有效手段修复损伤皮肤及其附属器（汗腺等）成为当前创面修复研究的新方向，目前尚缺乏相关的系统研究。脐带间充质干细胞（hUC-MSCs）以其易获取性、低免疫原性及多向分化潜能成为汗腺再生研究新的理想干细胞源。通过前期研究发现，hUC-MSCs经特定微环境诱导后具有向汗腺分化的潜能。本研究将通过分析现有汗腺发育基因在hUC-MSCs向汗腺分化过程中的调控作用，筛选出可用于构建汗腺分化微环境的基因。探索利用特定基因构建成品化汗腺分化诱导微环境，并将其用于诱导hUC-MSCs向汗腺细胞分化，继而评估此诱导体系获取的汗腺样hUC-MSCs参与早期修复与重建损伤汗腺组织的效能，并为构建含汗腺组织工程皮肤提供一种新的种子细胞源。

本项目主要立足于重度大面积烧伤患者损伤汗腺(SGs)排汗障碍的研究背景，严重损伤的汗腺组织无法依靠自身的修复潜能达到再生汗腺的目标。本项目的研究内容着眼于利用热休克汗腺上清液构成的汗腺分化诱导培养基培养人脐带间充质干细胞(hUC-MSCs)，从中筛选出具有诱导效能的汗腺发育基因角化细胞生长因子KGF；利用重组人角化细胞生长因子rhKGF构建的新型汗腺分化诱导培养基诱导hUC-MSCs分化为汗腺样细胞(SGCs)，并将获取的SGCs用于汗腺损伤的动物模型的治疗、进一步评价其修复效果。. 本项目研究中，以未分化hUC-MSCs为阴性对照，以正常汗腺细胞为阳性对照，通过将不同体积分数的热休克汗腺上清液与普通汗腺细胞培养基混合后构成不同浓度的汗腺分化诱导培养基，结果发现，以热休克汗腺上清液体积分数为20%的汗腺分化诱导培养基促进hUC-MSCs向SGCs分化诱导的效率最高。并对分化诱导过程中上清液标本和SGCs样本进行检测发现，汗腺发育基因表皮生长因子EGF、角化细胞生长因子（KGF）在此分化诱导过程中的表达效能最高。并且利用重组人角化细胞生长因子生长因子（rhKGF）复合普通汗腺培养基构成新的汗腺分化诱导培养基，并且成功诱导hUC-MSCs分化为SGCs。同时利用烫伤重度免疫缺陷鼠（SCID，即裸鼠）脚掌的方式，构建汗腺损伤动物模型。然后将获取的SGCs采用原位移植的方式移植于损伤脚掌部位；观察21天，待创面完全愈合后，取 脚掌组织作为标本，检测损伤部位组织表达汗腺标记物抗原：癌胚抗原CEA、细胞角蛋白CK14、CK19。结果发现，无SGCs移植的创面无汗腺标记物抗原的阳性表达，而SGCs移植侧创面可见组织中散在分布有汗腺标记物抗原阳性的组织。基于本项目的研究结果，可以构建出一种新的汗腺分化诱导培养基，为后续的间充质干细胞向汗腺样细胞分化提供一种较成熟的分化诱导途径，也为后续利用汗腺发育基因KGF构建成品化的汗腺分化诱导培养基奠定一定的研究基础。hUC-MSCs分化诱导获得SGCs的方式，也为干细胞在汗腺再生的研究领域提供了一种新的种子细胞，为后续利用干细胞大面积修复损伤汗腺研究中奠定了坚实的细胞学基础。本研究中，利用hUC-MSCs来源的SGCs修复损伤汗腺组织，其研究结果表明利用hUC-MSCs修复损伤汗腺具有较大的研究前景。