miR-141调控OP小鼠BMSCs成骨分化机制及健骨颗粒干预作用研究

Postmenopausal osteoporosis (PMOP) is a common disease in the elderly. Because of its unknown pathogenesis and poor clinical effectiveness, PMOP becomes a worldwide health problem. The reduction of Bone formation is the key factor of PMOP. For preventing reduction of Bone formation, enough Osteoblasts are the crux. Since the bone marrow mesenchymal stem cells (BMSCs) are considered to be the main source of osteogenic precursor cells in the bone remodeling, whether the differentiation and maturation of osteoblasts successfully happened from the BMSCs is the prerequisite of bone formation. Osteogenic differentiation of BMSCs has a closed relationship with micro RNA (miRNA). On the basis of related researches and pre-tests, this study was designed to investigate the regulatory role of MiR-141 and its target gene DLx5 in osteogenic differentiation of BMSCs. This study was designed via vivo and vitro by using Real Time RT-PCR, Western Blot, bioinformatics, and cell transfection. The purpose of the study is to reveal the pathogenesis of PMOP from the aspect of miRNA and to clarify the preventive mechanism of Jiangu granule on PMOP, providing new ideas about preventing postmenopausal osteoporosis by traditional Chinese medicine.

绝经后骨质疏松症（PMOP）是中老年人常见多发病，因其发病机理未明，临床疗效亦不尽人意，成为当今世界性健康难题。骨形成减少是PMOP发生的关键环节，而成骨细胞是骨形成关键细胞，骨髓间充质干细胞（BMSCs）被认为是骨重建过程成骨前体细胞主要来源，故成骨细胞是否顺利从BMSC分化成熟是决定骨形成的先决条件。微小RNA（miRNA）与BMSCs成骨分化关系密切。在完成前期相关研究及预实验基础上，本课题通过在体和体外实验，运用Real Time RT-PCR、Western Blot、生物信息学、细胞转染等技术手段，逐层深入探讨卵巢摘除后骨质疏松症形成过程中miR-141及其成骨靶基因DLx5 对BMSCs成骨分化的调控作用，从miRNA角度揭示PMOP发病机制，并进一步阐明中药健骨颗粒的可能作用机理，为中医药防治绝经后骨质疏松症提供新思路。

绝经后骨质疏松症（PMOP）因其发病率高、病因未明、疗效不尽如人意成为当今世界性健康难题，骨髓间充质干细胞（BMSCs）成骨分化能力是决定骨形成的先决条件，微小RNA（miRNA）与BMSCs成骨分化关系密切，而前期研究提示补肾健脾中药能明显促进骨形成。因此，本课题通过在体和体外实验，运用Real Time RT-PCR、Western Blot、生物信息学、细胞转染等技术手段，逐层深入探讨卵巢摘除后骨质疏松症形成过程中miR-141及其成骨靶基因DLx5 对BMSCs成骨分化的调控作用，从miRNA角度揭示PMOP发病机制，并进一步阐明补肾健脾中药健骨颗粒的作用机理。研究结果显示：去卵巢组BMSCs miR-141表达量明显高于假手术组（P﹤0.01），说明：卵巢切除雌激素水平下降对BMSCs miR-141的内源性表达产生明显影响，明确了miR-141与PMOP的密切关系。通过网站预测以及荧光素酶表达，证实了miR-141与Dlx5之间的靶基因关系。通过细胞转染、Western-Blot验证显示：miR-141对Dlx5/Msx2/Runx2级联反应具有负性调控作用，即：雌激素水平下降后，内源性miR-141表达升高，作用于Dlx5/Msx2基因，影响成骨核心因子Runx2表达，从而干预BMSCs的成骨分化过程。通过动物在体实验（中药灌胃）和体外细胞实验（含药血清），结果显示：补肾健脾中药能降低卵巢切除后miR-141 mRNA和蛋白水平，通过调控信号通路Dlx5/Msx2/Runx2提高Dlx5、Runx2等成骨关键因子的表达，增强BMSCs成骨分化能力，促进骨形成。miR-141/Dlx5/msx2/Runx2级联调控通路是补肾健脾中药防治PMOP的重要途径之一。本研究结果对揭示PMOP发病机制，阐明补肾健脾中药作用机制具有积极的科学意义和学术价值。