miR-203调控诱导多能干细胞向表皮定向分化作用与机制研究

Recent studies indicate that patient-specific or disease-specific induced pluripotent stem cells (iPSCs) can be obtained through the induction of pluripotent stem cells technology which have the same source of genetic background without immune rejection and ethical restrictions. Meanwhile, iPSCs are very similar to embryonic stem cells on clone morphology, growth characteristics, surface markers, gene expression patterns, epigenetic characteristics and vitro differentiation, which provide a vast source of donor cells for its application in the regenerative tissue repair. This study intends to induce the fibroblasts from the skin of burn patients to generate iPS cells by importing of plasmid using a electroporation method,thus to establish individual-specific non-integrative iPSCs lines and focuses on its directed differentiation capacity modulated by miR-203 towords epidermal stratification, the related molecular alteration of p63 and Wnt pathway, and its epigenetic modification mechanisms including DNA methylation, histone acetylation and methylation, and microRNA during the differentiation, and its effects to wound healing, which will lay an experimental foundation for the application of individual-specific iPSCs in the construction of skin tissue engineering and wound regenerative repair.

近期研究表明，通过诱导多能干细胞（iPS细胞）技术可获得与其来源遗传背景完全相同、不受免疫排斥和伦理学等限制的患者或疾病特异性iPS细胞，且在克隆形态、生长特性、表面标志物、基因表达模式、表观遗传特征及体外分化等方面与胚胎干细胞非常相似，为其在再生组织修复中的应用提供了广阔的供体细胞来源。已有研究和本课题前期实验均证实，模拟皮肤发育微环境可诱导iPS细胞定向分化为表皮或表皮细胞，但诱导效率很低；miR-203是表皮发育的决定因子和开关。本项研究拟在质粒电转法诱导烧伤患者皮肤来源的成纤维细胞生成iPS细胞、建立个体特异性无整合iPS细胞系基础上，系统观察miR-203调控iPS细胞向表皮定向分化作用及其分化过程中p63、Wnt通路相关分子改变及miRNA、DNA甲基化、组蛋白修饰等表观遗传学机制变化和对创面修复效应，为个体特异性iPS细胞在皮肤组织工程构建和创面再生修复中的应用奠定实验基础。

采用组织工程技术构建人工皮肤是修复大面积皮肤缺损创面的重要策略，而种子细胞的来源不足成为制约该项技术临床推广应用的瓶颈难题。本项目采用组织块贴壁法获得烧伤患者皮肤来源的成纤维细胞，采用不干扰基因组功能的非整合仙台病毒载体转染烧伤患者皮肤来源成纤维细胞，成功构建烧伤患者个体特异性iPSCs。重编程得到的iPSCs胚胎干细胞标志物Oct4、Sox2、Nanog、TRA-1-60、TRA-1-81、SSEA4表达均呈阳性；核型分析显示二倍体染色体数目为2N=46，XY，染色体正常；体外悬浮培养分化形成拟胚体；iPSCs中Oct4基因表达阳性，而在EB细胞中GATA4、SOX1、MSX1表达阳性，证实iPSCs在体外培养条件下具有分化成三个胚层的能力；重编程得到的iPSCs体内分化形成畸胎瘤，同样具有分化成三个胚层的能力；OCT4和NANOG的启动子区域甲基化状态检测显示在iPSCs中呈现高度去甲基化。结果证实重编程得到的iPSCs与其来源遗传背景相同，不受免疫排斥和伦理学等限制，在克隆形态、生长特性、表面标志物、基因表达模式及体内外分化等方面与胚胎干细胞非常相似，有望为其在皮肤再生修复中的应用提供广阔的供体细胞来源。在此基础上，系统观察了MiR-203调控iPS细胞向表皮定向分化作用及其分化过程中相关分子改变及microRNA等表观遗传学机制变化和对创面修复效应，为个体特异性iPS细胞在皮肤组织工程构建和创面再生修复中的应用奠定了实验基础。