FoxA2通过Gli信号调控干细胞成髓核分化的作用及机制研究

Disc degenerative disease has a high prevalence, and causes great burden of patients and society. Stem cell therapy is a research hotspot in the treatment of disc degeneration nowadays. However, the specific methods of stem cell differentiation towards nucleus pulposus cell type have not yet been developed. Recent studies have demonstrated that extracellular matrix regulates the differentiation of stem cells. Our previous studies found that collagen type II, which is the main extracellular matrix of nucleus pulposus in healthy intervertebral disc, had the ability to differentiate mesenchymal stem cell towards nucleus pulposus cell type. Moreover, the expression of transcription factor forkhead box protein A2 (FoxA2), the essential nucleus pulposus development factor, had been up-regulated in adipose-derived mesenchymal stem cells after collagen type II treatment, and FoxA2 regulated the expression of Gli family members, which involved in the crosstalk of Shh and Wnt/β-catenin signaling. As a result, we hypothesized that FoxA2 might regulate the differentiation process through Gli signaling. In this study, via molecular biology, cell biology and in vivo experiments, the mechanism of FoxA2 up-regulation by collagen type II in adipose-derived mesenchymal stem cells will been studied, the manner and mechanism of FoxA2 regulating Gli downstream Shh and Wnt/β-catenin signaling to determine differentiation towards nucleus pulposus cell type will been investigated, and the repair effect of differentiated stem cells and FoxA2 related mechanism will been verified in vivo. This study will facilitate the development of specific methods of differentiation towards nucleus pulposus cell type in vivo, and provide experimental and theoretical basis for the treatment of degenerative disc disease.

椎间盘退行性疾病患病率高，对患者和社会造成巨大的负担。干细胞治疗是现今退变椎间盘治疗的研究热点，但目前缺乏有效的体内诱导干细胞成髓核分化的方法。近期研究表明细胞外基质对干细胞的分化有调控作用。我们前期工作重要发现，正常髓核的主要细胞外基质Ⅱ型胶原作为细胞外支架具有诱导脂肪干细胞成髓核分化作用。Ⅱ型胶原能上调髓核发育的重要因子FoxA2，且该因子可调控Shh和Wnt通路交互作用关键因子Gli的表达。据此我们提出假说：FoxA2通过Gli信号介导Ⅱ型胶原诱导干细胞成髓核分化过程。本课题拟进一步研究Ⅱ型胶原诱导脂肪干细胞成髓核分化中FoxA2的作用，探索FoxA2调控Gli的方式及其通过Gli信号调节Shh和Wnt通路决定干细胞成髓核分化的关键机制，阐明Ⅱ型胶原荷载脂肪干细胞对退变椎间盘的修复效果。本研究将提出一种安全有效的体内髓核分化的诱导方法，为椎间盘的干细胞转化治疗提供实验依据和理论基础。

椎间盘退行性疾病患病率高，对患者和社会造成巨大的负担。干细胞治疗是现今退变椎间盘治疗的研究热点，但目前缺乏有效的体内诱导干细胞成髓核分化的方法。前期研究表明细胞外基质对干细胞的分化有调控作用。通过本项目的研究发现，正常髓核的主要细胞外基质Ⅱ型胶原作为细胞外支架具有诱导脂肪干细胞成髓核分化作用。且II型胶原诱导脂肪干细胞成髓核细胞分化与FoxA2介导的Shh信号通路激活相关。过表达FoxA2显著提升髓核特异性基因的表达与细胞外基质的合成，敲降FoxA2后脂肪干细胞成髓核细胞分化效率降低，Shh信号通路激活受抑制。Shh信号通路抑制剂GDC-0449可部分逆转过表达FoxA2所引起的成髓核分化。此外，II型胶原诱导的脂肪干细胞成髓核分化与FoxA2抑制整合素受体α2表达相关，而整合素受体α2的抑制又可进一步促进脂肪干细胞成髓核细胞分化。同时，体内研究结果表明，II型胶原支架荷载的脂肪干细胞能够有效修复退变椎间盘。上述研究结果证实，II型胶原通过上调FoxA2的表达激活Shh信号通路从而促进脂肪干细胞成髓核细胞定向分化，抑制整合素受体α2有利于FoxA2表达与Shh信号通路激活。明确了Ⅱ型胶原荷载脂肪干细胞对退变椎间盘的修复效果。本项目提出一种安全有效的体内髓核分化的诱导方法，为椎间盘的干细胞转化治疗提供实验依据和理论基础。