补肾药通过TAZ与经典Wnt通路crosstalk调控肾虚型骨髓基质干细胞成骨能力的机制

Osteoporosis is caused due to the process of bone remodeling, which loss of balance between bone formation and bone resorption leading to loss of total bone mass and is closely related to the differentiation of osteoblasts (OB). The Hippo pathway affects cell proliferation,differentiation,and the process of apoptosis in mamals vivo.Studies have shown that,Hippo downstream target gene transcription coactivator TAZ is able to respectively promote the differentiation of bone marrow stromal cells (BMSCs) to OB, regulate the OB expression of the specific gene osteocalcin,regulate the growth of bone and kidney,control Wnt/β-catenin (Wnt) pathway the function of transcription reaction.Wnt pathway plays an important role in the OB differentiation,mature.However,there is no clear report about whether crosstalk between TAZ and Wnt pathway can affect the osteogenic ability of BMSCs and tonifying kidney herbal can improve the activity of TAZ.For this reason,this research has two parts,one is to apply the overexpression and siRNA recombinant adenovirus transfection technology to reseach TAZ and Wnt pathway crosstalk effect and mechanism in the osteogenic ability of BMSCs,the other is to study whether the mechanism of the tonifying kidney herbal affecting the osteogenic ability of Kidney-deficiency BMSCs is related to TAZ and its regulation of the Wnt pathway,and from the view of molecular to clarify that the Wnt pathway,which is regulated by TAZ,is essential exemplification about "the kidney generating marrow and dominating bone".

骨质疏松症是由于骨重建过程中骨形成和骨吸收失平衡导致骨总量丢失所致，与成骨细胞（OB）分化密切相关。Hippo通路影响着哺乳动物体内细胞增殖、分化和凋亡过程。研究表明，Hippo下游的靶基因转录共激活因子TAZ具有促进骨髓基质干细胞（BMSCs）向OB分化，调节OB特异基因骨钙素表达，调节骨、肾发育，调控Wnt/β-catenin（简称Wnt）通路转录反应的功能。Wnt通路在OB分化、成熟中扮演重要角色。但是，对TAZ与Wnt通路crosstalk是否影响BMSCs成骨能力以及补肾药能否提高TAZ活性尚无明确报道。本课题①拟采用超表达和siRNA重组腺病毒转染技术研究TAZ与Wnt通路crosstalk对BMSCs成骨能力的影响及其作用机理；②研究补肾药对肾虚型BMSCs成骨能力的影响机制是否与TAZ及其调控的Wnt通路有关，并从分子角度阐明TAZ调控的Wnt通路是"肾主骨生髓"实质体现。

（1）成人骨髓间充质干细胞相关研究.研究观察成人骨髓间充质干细胞（hBMSCs）成骨诱导分化过程中芳香化酶(AROM)和雌激素相关受体(ER α、ERβ和ERRα)的表达。将细胞分为单纯培养组和成骨诱导组，MTT检测细胞增殖能力，茜苏红染色观察成骨矿化能力，qPCR和Western-Blotting检测AROM、ERα、ERβ 和ERRα mRNA和蛋白表达，ELISA检测细胞上清液和裂解液中雌二醇水平。结果发现hBMSCs及成骨诱导分化后能表达AROM、ERα、ERβ和ERRα，亦能自身合成和分泌雌激素，由此产生的雌激素可能是通过相关受体对hBMSCs成骨分化发挥作用。进一步观察雌激素缺乏型hBMSCs的肾虚表型特点。结果发现高浓度来曲唑(10-7 mol/L)在成骨诱导环境中能明显抑制hBMSCs增殖及向成骨分化，同时降低hBMSCs内cAMP表达及合成和分泌雌二醇的能力，说明采用来曲唑抑制AROM介导的雌激素合成途径的hBMSCs具有肾虚表型特点。研究结果《芳香化酶和雌激素受体在成人骨髓间充质干细胞中的表达》、《雌激素缺乏型人骨髓间充质干细胞的肾虚表型》在《中国组织工程研究》上发表。.（2）大鼠骨髓间充质干细胞相关研究.在大鼠BMSCs实验中，我们研究了《补肾中药淫羊藿苷促进BMSCs成骨分化的机制》，实验分为4组，即单纯BMSCs组、BMSCs+淫羊藿苷组、BMSCs+ICI-182780+淫羊藿苷组、BMSCs+DKK1+淫羊藿苷组，4组均在成骨诱导剂作用下培养。结果发现淫羊藿苷作用于BMSCs能明显促进TAZ、β-catenin蛋白的表达、抑制p-TAZ-Ser89、p-β-catenin蛋白的表达，而提前加以雌激素受体抑制剂ICI-182780、DKK1预处理，能明显抑制淫羊藿苷的这种作用。进一步开展《中药对骨髓间充质干细胞迁移的调控机制研究》，研究了虎杖苷对BMSCs迁移的调控机制，结果发现30μM虎杖苷明显促进BMSCs迁移。研究结果已被SCI 收录（Wei QS. Biomedicine & Pharmacotherapy, 2016）、（Wei QS. Biomedicine & Pharmacotherapy, 2017）、（Chen ZQ, Wei QS. Biomedicine & Pharmacotherapy, 2016）。