人毛囊干细胞成骨转分化的分子机制研究及骨缺损修复

The properties of hair follicle stem cells (HFSCs) in its readily accessibility and great proliferation potential make it an attractive cell source superior to adult mesenchymal stem cells in tissue engineering and cell therapy. However, up to now, no report has been found in regeneration of mesoderm derived tissue such as bone by the ectoderm derived HFSCs. Epithelial-mesenchymal transition (EMT) was defined by the formation of mesenchymal cells from epithelia by loss of their epithelia markers. EMT in keratinocyte plays crucial roles in skin development by formation of dermal and peri-follicle mesenchymal cells, as well as in adult wound repair, through a variety of mechanisms in which inhibition of P63 play a key role as indicated by previous studies. We found that, in our own studies, in vitro cultured HFSCs acquired an mesenchymal phenotype with treatment by inflammatory cytokines including TNF-α and IL-1β. Expression of P63 was decreased with occurance of EMT. Furthermore, several microRNAs such as miR130a, miR203, miR27a and miR106b was identified with inhibition of P63. More importantly, we found that HFSCs through EMT process could be induced to transdifferentiate into osteogenic lineage in vitro. Thus, purpose of this study is to investigate how to induce HFSCs, which has been manipulated to adopt a EMT pathway, to transdifferentiate into osteogenic mesenchymal cells in construction of tissue engineered bone to repair cranial bone defect. Moreover, epigenetic mechanisms responsible for mesenchymal transition of HFSCs was needed further studies in this project.

表皮毛囊干细胞(HFSCs)取材简单，扩增能力远远大于中胚层来源的间质类干细胞（如骨髓基质干细胞），但至今尚未有应用于中胚层组织如骨缺损的研究报道。上皮－间质转换(EMT)是指上皮细胞通过特定程序转化为具有间质表型的生物学过程。毛囊与表皮细胞通过EMT，参与了真皮与毛囊周间质组织的发生发育及皮肤创面愈合过程，这一过程的核心是P63表达抑制。我们发现，炎性因子可诱导体外扩增的HFSCs发生EMT转换，伴有P63表达抑制，初步筛选出可能参与调控P63表达的若干microRNA；初步结果证实，发生EMT转换的HFSCs，可诱导表达部分成骨细胞表型。因此，本项目将进一步研究炎性因子促进HFSCs发生EMT转换、转换细胞成骨诱导的适宜条件，探讨调控这一过程的表观遗传学机制，最终实现应用转分化的HFSCs构建组织工程骨并体内修复骨缺损的研究目标。

毛囊干细胞和角质细胞扩增能力强，具有可塑性，是具有应用潜力的组织工程种子细胞，但至今尚未有应用于中胚层组织如骨缺损的研究报道。本课题成功探索并优化了角质细胞的分离和培养方法，并以该细胞为主成功进行了上皮-间质转化（EMT）诱导。定量PCR和Western blot技术证明，经TGFβ单独或与TNFα联合处理发生EMT转化的角质细胞上皮细胞标志分子E钙黏素明显下调，而间质细胞标志分子N钙黏素和波形蛋白表达显著上调。在TGFβ诱导的EMT过程中发现表皮细胞关键转录因子ΔNp63和上皮细胞重要microRNA—miR-203表达受到抑制，而调节EMT的重要转录因子ZEB表达明显上调。进一步对经EMT转化的角质细胞进行了成骨诱导并检测到部分成骨细胞表型，为接下来实现结合支架材料的组织工程骨体内修复奠定了基础。在本项目支持下，发表SCI论文6篇，累计影响因子达26分，培养博士研究生2名。