人成纤维细胞分化调控与皮肤及汗腺再生的基础研究

With extensive full-thickness burns or traumas, skin and its appendages like sweat glands can not regenerate to form their organization because of complete destruction of skin cells. Most survivors from severe burns or traumas face the problem of long-term loss of secretory function and endure great pain, which seriously affects patients' quality of life. Over the past century, remarkable progress has been achieved in restoring the structure of damaged and dysfunctional skin through auto-microskin or tissue-engineering technique and other methods, however, regenerating the function of damaged skin remains unsatisfactory. Therefore, the development of better treatment to enhance the regeneration of skin with normal functions will have a big impact on not only patients and their families but also the financial burden to health services. Recent breakthroughs in the research of cell reprogramming bring us new hope for skin regeneration. On comparing cell types from different sources, autologous dermal fibroblasts are thought to be the best choice for this purpose. First, abundant and easily accessible fibroblasts could be used. Such cells are easily isolated by punch biopsies from skin with minimal risk. They are cultured under plain conditions and have high potential for expansion in vitro. Second, one attractive feature of fibroblasts is its plasticity. Most recent studies suggest that reprogramming factors could induce fibroblast to dedifferentiate toward an unstable near-pluripotent state capable of differentiating along a specific lineage in the presence of permissive microenvironment, and that tissue-restricted transcription factors can dictate their fate across germ layer barriers, a hallmark of transdifferentiation. Nowadays, however, researches on induction of fibroblast to cells in skin and its appendages by these remarkable methods have not been reported. Therefore, based on our previous studies, we attempt to establish the dedifferentiation and transdifferentiation models of human dermal fibroblast to acquire epidermal stem cells and sweat gland cells by application of transcription factors and permissive microenvironments. These induced epidermal stem cells and sweat gland cells will be used to reconstruct a three-dimensional skin by organotypic cultures. We will further test the availability of these induced cells or their generated skin equivalent in in vivo grafting models and determine their involvement in reconstituting normal skin and its appendages. This research is original and novel in theory. It will be a new part of importance in the study of cellular differentiation and reprogramming. Moreover, the development of methods for the efficient induction of fibroblasts to the two critical cells for the structural and functional regeneration of skin will enable us to fulfill the skin's perfect regeneration. Outcomes from this study will hopefully pave a good foundation for the exploration of in situ skin regeneration.

全层大面积创伤使皮肤及汗腺等附件完全损毁且难以再生，皮肤功能丧失，患者生活质量受到严重影响。如何获取足够数量皮肤细胞（如表皮干细胞和汗腺细胞）重建具备完整结构和功能的皮肤是皮肤及其附件再生医学研究中凝炼出的重要科学问题。成纤维细胞具有获取皮肤细胞构建完整皮肤的优势：一是来源丰富。成纤维细胞可从创伤部位获取，易于体外培养和大规模扩增；二是具有可塑性。在特定因素调控下成纤维细胞可去分化或转分化为多种干细胞和成体细胞。本研究中，我们将在前期工作基础上，采用转录因子诱导和微环境干预的方式建立人皮肤成纤维细胞去分化和转分化模型，获得诱导性表皮干细胞和汗腺细胞并构建全层皮肤组织，通过体内损伤模型验证其参与修复与再生的能力。该研究在理论上具有一定原创性并将进一步丰富成体细胞诱导分化理论；同时在实际应用中有可能建立起在皮肤严重损伤条件下促进皮肤及汗腺再生的实用技术和方法，为真正实现皮肤的完美修复奠定基础。

1. 完成的研究内容.(1) 完成人真皮成纤维细胞体外分离、培养、纯化和Luciferase标记。利用重编程慢病毒载体转染成纤维细胞，结合专用表皮干细胞培养基，成功将成纤维细胞诱导成表皮干细胞样细胞。.(2) 通过研究胎儿皮肤汗腺发育过程中基因表达的变化，筛选出汗腺发育相关基因(EDA和FGF10)。构建EDA和FGF10基因的慢病毒载体，随后感染人真皮成纤维细胞，诱导其变成汗腺样细胞。.(3) 利用诱导的表皮干细胞样细胞和汗腺样细胞在体外构建成三维皮肤组织，检测皮肤细胞的生长情况以及复层表皮和汗腺等皮肤附件形成情况。.(4) 将体外构建三维皮肤组织移植到全层皮肤缺损动物模型中，评估体外构建的皮肤组织在体内参与损伤修复和再生能力。.(5) 将诱导的表皮干细胞样细胞和汗腺样细胞移植到全层皮肤损伤动物模型中，评估诱导的表皮干细胞样细胞和汗腺样细胞直接移植参与皮肤组织损伤修复和再生的能力。..2. 取得研究成果.(1) 我们建立了新的诱导人真皮成纤维细胞重编程为表皮干细胞的模型。该诱导模型为诱导其他功能细胞提供了一种便捷思路，即将重编程因子和适宜的培养环境进行结合，可以获取大量所需的种子细胞，极大地简化了基于转录因子的重编程策略时筛选关键转录因子的繁琐过程。.(2) 利用特定基因将成纤维细胞诱导成为汗腺样细胞。汗腺修复和再生是烧（创）伤领域的科学难题，其中获取足够数量的汗腺细胞是关键。该诱导模型为获取一定数量并可用于移植的汗腺细胞提供了可靠来源。.(3) 验证了诱导的表皮干细胞样细胞和汗腺样细胞参与皮肤损伤修复和再生的能力。利用诱导的表皮干细胞样细胞和汗腺样细胞体外构建出完整的三维皮肤，并在全层皮肤损伤动物模型上进一步证实了构建的皮肤组织参与皮肤修复和再生的能力。这为体外构建人工三维皮肤提供了实用技术。.(4) 验证了诱导的表皮干细胞样细胞和汗腺样细胞直接通过细胞移植参与皮肤损伤修复和再生的能力，这为通过移植诱导细胞在损伤部位实现皮肤组织实现原位再生奠定了夯实的基础。