II型胶原在脂肪干细胞成髓核细胞定向分化中对转录因子FoxA的调控机制及修复退变椎间盘的研究

Stem cell committed differentiation is the key to the success of disc biological regeneration by stem cell transplantation. Previous studies have shown the role of various growth factors in stem cell differentiation and its certain regeneration effect of disc degeneration. However, growth factors still have lack of effective regulation for stem cell advanced differentiation into nucleus pulposus cell, and restrict the regeneration effect of stem cell transplantation. Recent studies have shown that FoxA/Shh signaling pathway has important implications for the differentiation of stem cells into nucleus pulposus cells and mainly involves the regulation of advanced committed differentiation, which has been less affected by most commonly used growth factors. Our previous studies demonstrated that type II collagen differentiated adipose-derived stem cells (ADSCs) into nucleus pulposus cells (NPCs), and could improve FoxA expression, but the mechanism remains unknown. We considered that type II collagen could regulate FoxA expression through binding with integrin, which was different from TGF regulation targets, moreover, both of II collagen and TGF may have a synergistic effect to induce committed differentiation. This subject aims to study the mechanism of transcription factor FoxA regulation by type II collagen in committed differentiation from ADSCs to NPCs, and the synergetic effect of inducing committed differentiation of type II collagen and TGF-β3, in order to established theoretical basis of two-step method of committed differentiation from ADSCs to NPCs.

干细胞定向分化是干细胞移植生物学修复退变椎间盘成功的关键。目前的研究已显示各种生长因子具有诱导干细胞分化的作用，对退变椎间盘具有一定的生物修复效果。然而，生长因子对干细胞成髓核细胞的中晚期分化缺乏有效的定向调控仍制约了干细胞发挥有效的作用。近来的研究显示FoxA/Shh信号通路对干细胞成髓核细胞分化具有重要的意义，且主要涉及中晚期定向分化的调控，但目前常用的生长因子对其无明显作用。我们前期的研究结果显示Ⅱ型胶原能诱导脂肪干细胞成髓核细胞分化且能提高FoxA表达，但机理仍不清楚。我们认为Ⅱ型胶原可能通过与整合素结合而发挥对FoxA的调控，与TGF调控属于不同的作用靶点，两者可能具有诱导定向分化的协同效应。本课题旨在通过研究诱导脂肪干细胞成髓核细胞定向分化过程中Ⅱ型胶原对转录因子FoxA调控的机制及其与TGF-β诱导分化的协同作用，为创建脂肪干细胞定向分化为髓核细胞的双阶段调控法奠定理论基础。

干细胞定向分化是干细胞移植生物学修复退变椎间盘成功的关键。目前的研究已显示各种生长因子具有诱导干细胞分化的作用，对退变椎间盘具有一定的生物修复效果。然而，生长因子对干细胞成髓核细胞的中晚期分化缺乏有效的定向调控仍制约了干细胞发挥有效的作用。本研究于体外通过信号通路阻滞剂、siRNA沉默技术及基因转染技术，明确了II型胶原诱导ADSCs成髓核细胞定向分化的作用及调控转录因子FoxA2，明确其定向分化的关键靶点，为提高ADSCs成髓核细胞分化的效率提供理论依据；研究了II型胶原和生长因子诱导干细胞分化的协同作用，提高ADSCs定向分化能力，创建双阶段法诱导ADSCs成髓核细胞定向分化；在此基础上，于已创建的椎间盘退变模型中验证了成髓核细胞定向分化的ADSCs的生物学修复效果。本研究结果证实II型胶原具有特异性诱导ADSCs成髓核细胞定向分化的作用，且调控髓核细胞分化相关转录因子FoxA2。通过II型胶原与生长因子cocktail（TGF-β3+IGF-1）诱导分化的协同作用可显著提高ADSCs成髓核细胞定向分化能力。通过本研究，为创建脂肪干细胞定向分化为髓核细胞的双阶段调控法奠定理论基础和实验依据，为椎间盘退变的生物学治疗提供了新的思路。