N-乙酰氨基葡萄糖转移酶V对间充质干细胞迁移、分化的作用及分子机制研究

Messenchymal stem cells (MSCs) are a type of adult stem cells and have advantages of immune regulation and multi-differentiation. MSCs have been used as ideal seed cells for tissue engineering and regenerative medicine, while the characteristic of MSCs migrating to ischemic or inflammatory tissue and the efficiency of differentiation are the keys to safe and effective clinical application. Many glycoproteins, including cell-surface proteins, ECM components, contribute to cell migration and differentiation. The content and branches of oligosaccharides on glycoproteins, may play critical roles in regulating glycoproteins function and cell migration and differentiation. Thus, N-acetylglucosaminyltransferase V (GnT-V), a key enzyme in the formation of branching of asparaginelinked oligosaccharides, which synthesize oligosaccharide are always considered as important factors regulating cell migration and differentiation. In many type cells, GnT-V expression has been shown to be related to migration and differentiation potential via glycosylated glycoproteins, such as integrins, growth factor receptors, and TrKA receptor. Whereas, in MSCs, the mechanism of GnT-V roles in cell migration and differentiation is still not fully understood. Therefore, the aim of this thesis to clarify the biological function of GnT-V in MSCs and its molecular mechanism, trying to find key protein targets and strategies intervening even increasing cell migration and differentiation of MSCs. And provide the oretical bais and effective transplantation method more effectively in clinic for application of MSCs.

间充质干细胞（MSCs）因易获取、免疫原性小、多向分化等优点被作为再生医学的理想种子细胞。更高效的迁移到病变组织并定向分化是MSCs安全有效的临床应用关键。MSCs迁移、分化作用是细胞糖蛋白与环境作用结果，N-聚糖结构改变影响糖蛋白功能，进而影响细胞迁移、分化，提示糖基转移酶可作为调节因子。N-乙酰氨基葡萄糖基转移酶V(GnT-V)催化合成的β1,6分支N-聚糖是形成复杂型N-聚糖限速酶，在胚胎发育、组织修复等过程中作用重要。研究表明，GnT-V通过增加细胞表面糖蛋白β1,6分支N-聚糖影响细胞迁移能力；MSCs成骨分化伴随β1,6分支N-聚糖增加； GnT-V增加TrKA 受体N-聚糖β1,6分支促进神经干细胞分化。若证实GnT-V及产物N-聚糖β1,6分支参与MSCs迁移、分化，阐明调控机制，寻找增强MSCs迁移、分化能力的关键分子，将为MSCs在组织修复再生医学应用提供新靶点和思路。

间充质干细胞（MSCs）作为一种多潜能干细胞，存在于多种组织中，在组织损伤修复中发挥重要作用。MSCs迁移、分化潜能实质上是MSCs与周围环境之间依靠细胞表面分子相互作用的结果。GnT-V通过增加细胞表面整合蛋白、钙粘蛋白、生长因子受体的N-聚糖β1,6分支结构而增加其蛋白质稳定性及信号传导发生改变增强细胞的迁移能力，而对MSCs分化调控有待研究。.我们发现在MSCs成骨分化过程中，细胞表面三、四天线型的N-聚糖数量增加，即β1,6分支N-聚糖结构表达过量。说明MSCs分化能力与GnT-V及其产物N-聚糖的改变密切相关。为了研究GnT-V在MSCs分化中的作用，构建了GnT-V敲低及过表达的细胞稳定珠，发现下调GnT-V后表现为抑制MSCs成骨分化作用；相反的过表达GnT-V的MSCs促进成骨分化。证明了GnT-V参与了MSCs成骨成脂分化过程中，作为成骨分化的正调控因素在机体的骨代谢中发挥重要的作用。.为了进一步阐明GnT-V在MSCs分化调控机制，检测了GnT-V过表达后PTPRT N-聚糖结构的增加致使其以二聚体的形式存在增多，单体减少导致其磷酸酶的活性减弱，我们进一步检测了其PTP家族的成员PTPN6（SHP1）,发现过表达GnT-V后SHP1的蛋白量增加，提示我们SHP1参与GnT-V调控MSCs分化作用。我们构建SHP1敲低及过表达MSCs稳转细胞株，发现SHP1表现为促进MSCs成骨分化。同时我们进一步用间充质干细胞特异性敲出SHP1小鼠模型也证明了SHP1缺失后影响了骨发育。.已有研究表明，Wnt/β-catenin信号通路参与调控MSCs分化过程。我们进一步研究GnT-V通过SHP1调控Wnt信号通路的方式，发现SHP1与GSK3β结合使pY216位点的去磷酸化，抑制了GSK3β的活性，β-catenin得到累积并入核，上调成骨分化相关转录因子的表达。反之， PTPN6(SHP1)的缺失，则会增强GSK3β对β-catenin的磷酸化降解作用，降低MSCs成骨分化能力。.综上所述，我们的研究揭示了GnT-V通过调控Wnt/β-catenin信号来维持MSCs的成骨成脂分化稳态，确保机体骨骼和脂肪组织的正常发育和功能的实现。这项工作首次揭示了GnT-V在MSCs分化中的作用，并且在临床上为干细胞治疗骨质疏松疾病。