锂剂通过β-catenin/Wnt调控内源性间充质干细胞预防/治疗激素型股骨头坏死的实验研究

There are currently no effective way to prevent or treat steroid induced osteonecrosis of femoral head (ONFH) at the early stage. The suppressed proliferating ability and the decreased number and the inbalanced differentiation of mesenchymal stem cells (MSCs) caused by steroid is one of the cellular mechanisms underlie.In our previous study, we showed for the first time that lithium, well known as an anti-depression drug, promoted proliferation of human bone marrow derived MSCs in vitro. Mechanism study revealed that this effects was dependent on the glycogen synthase kinase 3 beta (GSK3β) mediated canonical Wnt pathway activation. Lithium abolished dexamethasone's effect on suppressing MSCs proliferate, but the mechanism is still unclear. We thus hypothesize that steroid suppresses β-catenin/Wnt signaling pathway of mesenchymal stem cells and lead to osteonecrosis of the femoral head. Lithium suppresses GSK3β, which causes the accumulation of β-catenin and activation of Wnt pathway in vivo. Therefore, lithium may be effective for the prevention/early treatment of steroid induced osteonecrosis of femoral head. We will use a series of assays to confirm our hypothesis and further explore the mechanism. Firstly, we plan to up-regulate and down-regulate the expression of GSK3β and β-catenin to further investigate of lithium's protective role in vitro. Secondly, we will use a series of transgenic mice to confirm our findings regarding lithium's protective role. Three strains of transgenic mice will be involved in this study. They are Prx1-Cre/ERT-GFP mice, β-catenin-flox mice and β-cateninfx(Ex3)/fx(Ex3) mice. We will specifically activated the β-catenin gene in mesenchymal stem cells in adult mice by generating tamoxifen inducible β-catenin conditional activation mice through breeding of β-catenin fx(Ex3)/fx(Ex3) mice with Prx1-cre/ERT-GFP transgenic mice. We will use these mice to test whether steroid caused osteonecrosis of femoral head by down-regulating β-catenin. Meanwhile, We will generating tamoxifen inducible β-catenin conditional knock out mice through breeding of β-catenin-flox mice with Prx1-cre/ERT-GFP transgenic mice. These mice will be used to test whether lithium exerts its protective effect through up-regulating β-catenin. Finally, we plan to confirm the above findings in a classical rabbit steroid induced ONFH model, aiming to develop a novel way to prevent or treat steroid induced ONFH at early stage.

激素型股骨头坏死（ONFH）目前尚无法通过药物预防或早期治疗，局部间充质干细胞（MSCs）增殖能力下降及分化异常是重要的发病因素。我们发现锂剂可通过激活MSCs增殖及分化重要调控通路β-catenin/Wnt，促进体外MSCs增殖并拮抗激素的抑制作用。据此推测锂剂对激素型ONFH可能具有预防或治疗作用。本课题拟在体外继续探讨锂剂拮抗激素对MSCs分化的影响，通过蛋白过表达及SiRNA技术正负向调控β-catenin/Wnt通路活性，探究锂剂拮抗激素的分子机制；体内建立他莫昔芬可诱导性β-catenin-LoxP+/-转基因小鼠模型，特异性地在MSCs瞬时过表达或敲除β-catenin蛋白，探究锂剂调控内源性MSCs，拮抗激素预防ONFH的效果及分子学机制；最后在经典的兔激素型ONFH模型中验证锂剂疗效。从调控内源性MSCs增殖及分化入手，为临床预防/早期治疗激素型ONFH进行新的探索。

激素性股骨头坏死（ONFH）目前尚无法预防或极早期治疗，间充质干细胞（MSCs）增殖能力下降及数量降低是发病的重要因素。糖皮质激素通过抑制Wnt/β-catenin信号通路抑制骨髓间充质干细胞的成骨分化能力。移植外源性MSCs 虽可起到一定的修复作用，但仍存在滞后性。我们发现抗抑郁药锂剂可抑制GSK3β而激活骨修复重要调控通路β-catenin/Wnt，促进体外MSCs增殖，拮抗激素抑制作用。本课题研究 Wnt/β -catenin 信号通路体外实验锂剂预处理对高剂量激素环境下hBMSCs成骨方向分化的保护作用及验证动物实验。在体外通过GSK3β及β-catenin 蛋白过表达及SiRNA 技术正负向调控上述蛋白表达，探究锂剂拮抗激素抑制作用机制；体内研究中拟建立他莫昔芬可诱导性β-catenin-LoxP+/-转基因小鼠模型，特异性地在MSCs 瞬时过表达或敲除β-catenin 蛋白，在活体内探讨激素导致ONFH 的细胞学机制，锂剂促进内源性MSCs 增殖，拮抗激素作用的效果及分子学机制；最后在经典的兔激素型ONFH 模型中验证疗效。此外，在此研究基础上对实验做了补充，研究间充质干细胞对激素型股骨头坏死的病理机制进行了探讨，探索了滑膜间充质干细胞及富集血小板血浆来源的外泌体对激素性股骨头坏死的保护作用及其分子机制。本研究从激活内源性MSCs 增殖的角度入手，为临床预防/极早期治疗激素型ONFH进行新的探索。