KGF促进hUC-MSCs向汗腺分化的特定microRNA调控机制及其参与修复损伤汗腺的应用基础研究

The skin and its appendages could easily be destroyed by extensive and severe burns,which will lead to dysfunction of body temperature regulation and homeostasis maintenance. As a result, skin and its cutaneous appendages(sweat glands) lost its original structure and funtion to repair by itself. If we want to regenerate its original structure and function, we have to rely on various therapeutic technologies. To date, there were some effective ways for repairing damaged sweat glands, including transdifferentiation of bone marrow derived mesenchymal stem cells(BM-MSCs) into sweat glands through co-culturing with heat-shocked sweat gland cells(SGCs) or transfecting with specific development gene(ectodermal dysplasia gene,EDA). Among them, there would be a long time for proliferation and transdifferentiation of autologous BM-MSCs into SGCs, as well as fabrication of tissue engineering skin with mature SGCs. Based on our previous studies, we have found that mesenchymal stem cells derived fron human umbilical cord Wharton's jelly (hUC-MSCs) possessed many promising properties, including low immune-resistance, poly-differentiation potency, convenient isolation and proliferation.hUC-MSCs could also be induced to transdifferentiate into SGCs under specific induction micro-environment. In addition, Some sweat gland development genes have been found in the development of sweat glands.In this study, we will dwell on the mechanism of these development genes in the transdifferentiation into SGCs from hUC-MSCs.In addition,many microRNAs exhibit a temporal or tissue-specific expression pattern, indicating that they might have a crucial role in tissue and organ development, function and maintenance. We hope that we could find out the specific gene(s) and microRNA(s) which could play the important role in induction of hUC-MSCs into sweat gland-like cells. In latter stages, we will attempt to find out functional miRNAs related to keratinocyte growth factor(KGF) through microarray ananlysis,and find out the detailed mechanism of sweat gland-differentiation of hUC-MSCs. Meanwhile, we will verify the function of microRNAs in the process of transdifferentiation of sweat gland cells through ways of over-expressing and knock down of microRNAs.As a product, we would apply microRNA in promoting the efficacy of KGF in harvesting sweat gland-like cells in future,which could be used for repair and regeneration of damaged SGCs at the early period of wound healing. In a word, we hope to establish an efficient and biosafe differentiation system, as well as a technique for sweat gland regeneration, which supply us a potential therapeutic way to repair destroyed SGCs as early as possible.

重度大面积烧伤破坏人体皮肤结构与功能，导致患者体温调节与排汗功能缺失、生活质量下降。当前,利用自体BM-MSCs转分化潜能成为烧伤后期修复损伤汗腺的一种有效途径。如何在烧伤早期利用干细胞疗法完成皮肤及其附属器（汗腺等）的修复与重建成为皮肤再生研究的重要方向。人脐带间充质干细胞（hUC-MSCs）以其低免疫原性、易获取性及汗腺分化潜能成为汗腺再生研究的理想细胞。本项目以调控hUC-MSCs向汗腺定向分化的已知汗腺发育基因的筛选为基础，构建汗腺高效分化诱导体系。以关键发育基因提高hUC-MSCs汗腺分化效能为背景，从中筛选出汗腺分化体系中调控关键基因的特异microRNA及其分子作用机制，并对其调控活性进行双向评估。力求探索出一套利用关键microRNA增强基因表达、提高汗腺分化效能的理论依据与技术体系；并将其应用于由hUC-MSCs获取汗腺样细胞、及其参与早期修复损伤汗腺的效能与安全性研究。

大面积严重烧创伤后患者的创面愈合以及生活质量的提高成为目前烧创伤救治领域的重要难点。干细胞治疗学、组织工程技术（3-D打印技术）以及基因筛选工程的不断进展，为严重烧伤患者的组织修复与再生提供了先进的治疗技术。本项目研究的主要内容是创建新型的干细胞再生治疗损伤汗腺的技术体系，同时对于干细胞向汗腺样细胞（SGCs）分化的相关分子机制以及应用潜能进行深入探索。按照本项目研究的预期目标，经过前期研究发现：1.本项目研究采用的脐带间充质干细胞（hUC-MSCs）具有向SGCs分化的潜能；2.利用基因检测技术与发育生物学的相关生物信息学进行交叉研究，得出汗腺发育关键基因角化细胞生长因子的基因工程产物rhKGF具有诱导hUC-MSCs向SGCs分化的活动，并且在本项目研究中获得成品化的汗腺发育基因的诱导培养基。3.由本项目研究获得的SGCs具有修复、重建损伤汗腺的生物学活性。4.本项目研究中对涉及SGCs 分化的相关microRNA的相关机制进行初步探索，并且进行初步的功能验证。. 在本项目研究中，主要对诱导hUC-MSCs向SGCs分化诱导的培养基进行转化应用，申请了相关发明专利；在后续的研究中将采用本分化诱导体系对多种干细胞进行分化诱导效能的评价比较，最终得出更加具有高效分化诱导效能的干细胞。在本项目研究中获得的SGCs具有在体修复重建损伤汗腺的生物学活性；在后续的转化应用研究过程中，将采用组织工程技术与生物3D打印技术将SGCs修复重建损伤汗腺的活性剂效能逐步提高，为后续的干细胞转化应用潜能奠定基础。对于调控hUC-MSCs向SGCs分化的microRNA的基因筛选以及相关调控网络的机制研究，将为后续的分子作用机制研究提供更加坚实的理论基础。. 最终希望通过本项目研究及其后续的滚动支持逐步拓展损伤汗腺再生研究的基础理论研究的深度，同时希望通过本项目研究的相关研究成果将SGCs修复重建损伤汗腺组织的转化应用研究技术进行不断拓展；以期达到汗腺再生研究领域的基础研究与转化应用研究的完美结合。

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