胚胎真皮间充质干细胞诱导深Ⅱ度烧伤创面汗腺原位再生及稳态维持机制的实验研究

Human fetal skin wound can be healing scarlessly with the perfect regeneration of skin and eccrine sweat glands by EDA / EDAR signaling pathway, while human adult sweat glands structure can hardly regenerate after burn, and the lost function of perspiration greatly affect the life quality of patients. In order to promote adult human eccrine sweat glands regeneration and its homeostasis of microenvironment in deep second-degree burn wounds, on the basis of the preliminary research of human embryonic dermal MSCs（ED-MSCs）,we will, compared with BM-MSCs, (1)conduct the vitro sweat gland induction experiments of the ED-MSCs; (2)establish nude mouse model of deep second-degree burn in palms and nude mouse model transplanted adult human skin and create deep second-degree burn wound; (3)inject the BrdU-labeled ED-MSCs in the burn wounds. Through methodologies such as immunohistochemistry, in situ hybridization, flow cytometry, reverse transcriptase polymerase chain reaction (RT-PCR), western blot and other techniques, we will detect the differential expression levels of genes and proteins of EDA/EDAR pathway, regulating sweat glands regeneration, and the downstream NF-κB pathway. We will observe the sweat glands regeneration in situ, ED-MSCs phenotypic characteristics and its migration, orientation, phenotypic changes, and its effects on extracellular matrix composition, eccrine sweat gland morphogenesis, function reconstruction and wound healing. We will investigate molecular and cellular mechanisms of ED-MSCs on EDA gene activation, structure regeneration and homeostasis of sweat glands, and try to develop a new stem cell therapy to sweat glands regeneration in situ in deep burn wounds.

人胎儿皮肤损伤后通过EDA/EDAR信号通路实现汗腺等皮肤结构的完全再生，但成人深度烧伤后汗腺再生困难。为促进临床深Ⅱ度创面汗腺再生，维持局部微环境稳定，课题组在前期成功开展胚胎真皮间充质干细胞（ED-MSCs）研究的基础上，以成体BM-MSCs为对照，进行①ED-MSCs离体汗腺诱导实验；②建立裸鼠脚掌和成人皮肤深Ⅱ度烧伤创面模型；③ED-MSCs局部干预深Ⅱ度烧伤创面。应用细胞标记、免疫组化、逆转录聚合酶链反应和Western blot等技术，检测调控汗腺再生的EDA/EDAR通路及下游NF-κB通路基因及蛋白差异性表达水平，观察ED-MSCs在组织中的迁移、定位和表型变化，以及对ECM组成、汗腺形态发生和创面愈合的影响，综合分析创面局部ED-MSCs干预条件及其对汗腺上皮EDA基因活化、结构再生、功能重建及稳态维持的机制，为未来临床深度烧伤创面汗腺原位再生探索新的治疗途径。

人胎儿皮肤损伤后通过EDA/EDAR信号通路实现汗腺等皮肤结构的完全再生，但成人深度烧伤后汗腺再生困难。本课题组通过细胞标记、免疫组化、逆转录聚合酶链反应和Western blot等技术，检测调控汗腺再生的EDA/EDAR通路及下游NF-κB通路基因及蛋白差异性表达水平，综合分析创面局部FD-MSCs干预条件及其对汗腺上皮EDA基因活化、结构再生、功能重建及稳态维持的机制，为未来临床深度烧伤创面汗腺原位再生探索新的治疗途径。经过3年的探索研究，本课题组已成功构建多种动物模型，包括大鼠烧伤瘀滞区模型、小鼠深Ⅱ度烧伤创面模型、小鼠全层皮肤缺损创面模型、小鼠皮肤衰老模型、小鼠异体皮移植模型、裸鼠黑色素瘤模型等，为后续实验的开展奠定基础；成功提取胎儿真皮间充质干细胞（FD-MSCs）及其条件培养基和外泌体，并发现其对烧伤创面进展的抑制作用及血管内皮细胞的保护作用、对小鼠皮肤的抗衰老作用、对全层皮肤创面的促愈作用以及对小鼠黑色素瘤的抑制作用；探索并优化了DADM的获取方法，拓展DADM在烧伤治疗中的应用前景；成功制备负载间充质干细胞的生物纤维素膜并证实其对全层皮肤创面的促愈作用等。.以上成果说明了皮肤创面局部FD-MSCs干预条件对损伤修复和组织再生的调节机制，为创面完美修复提供新的理论、思路和实践依据，为进一步的科研计划奠定坚实的基础。