CHIR-99021与FGF8协同诱导人表皮干细胞牙向分化

Induction of Human Keratinocytes into Enamel-secreting Ameloblasts through the Synergy of CHIR-99021 and FGF8

人表皮干细胞(human keratinocyte stem cells,hKSCs)可作为上皮源性的成体干细胞应用于牙齿再生,但是其诱导效率较低.本研究利用小分子化合物CHIR-99021提高hKSCs的Wnt/β-catenin信号活性,再与具有诱导成牙潜能的小鼠牙胚间充质重组,构建嵌合体,并移植裸鼠肾囊膜下培养20 d.将嵌合体组织切片,并利用组织染色和免疫组化等方法鉴定牙齿结构.结果显示,经FGF8诱导处理的hKSCs与小鼠牙胚间充质构成的嵌合体的成牙率为27.80%,其中成釉率仅为40.00%;经CHIR-99021诱导处理的hKSCs与小鼠牙胚间充质构成的嵌合体的成牙率仅为18.20%,其中成釉率高达100%;而CHIR-99021与FGF8协同作用,则进一步提高嵌合体成牙率至40.00%,其中成釉率也达75.00%.进一步的研究发现,经CHIR-99021处理后,hKSCs的Wnt/β-catenin信号活性明显提高,同时FGF8的表达水平也显著上调.以上结果表明,CHIR-99021可通过上调Wnt/β-catenin信号活性水平,同时促进FGF8表达,与FGF8协同,高效诱导hKSCs分化为具有分泌釉质功能的成釉质细胞.研究结果对利用hKSCs作为上皮来源的成体细胞应用于人类牙齿再生的研究具有重要意义.

Human keratinocyte stem cells（hKSCs） can be used as epithelial adult stem cells in tooth regeneration. But the tooth formation rate from such cells is relatively low. In this study, hKSCs were treated with small molecules CHIR-99021 to activate the Wnt/β-catenin pathway before the hKSCs were recombined with mouse embryonic dental mesenchyme possessing odontogenic potential. Histology and immunohisto-chemistry staining were used to detect the chimeric tooth. The results showed that the efficiency of tooth formation（tooth formation rate） of the chimeric tooth treated with FGF8 was 27.80% , and the efficiency of hKSCs differentiation into ameloblasts（ameloblasts differentiation rate） was 40.00% among those formed teeth. CHIR-99021 could improve the ameloblast differentiation rate to 100%. The synergistic effect of CHIR-99021 and FGF8 could increase tooth formation rate to 40.00%, and ameloblast differentiation rate to 75.00%. Further research showed that CHIR-99021 could activate theWnt/β-catenin signaling for hKSCs, and increase protein and mRNA levels of FGFS. These results indicated that CHIR-99021 could activate the Wnt/β-catenin signaling for hKSCs and simultaneously enhance FGF8 expression, which increased the induction of hKSCs into ameloblasts. This study provides relevant experimental data for tooth regeneration which use hKSCs as a source of human epithelial stem cells.