Cx43/miRNA-206调控的成骨分化在激素性股骨头坏死中的作用及其机制研究

Steroid-induced osteonecrosis of the femoral head (SANFH) is a locally destructive disease with a multifactorial genesis. The majority of patients are men between 35 and 45 years of age, who are increasingly reduced in their quality of life and career. Recent studies have demonstrated that miR-206 was expressed in osteoblasts, and its expression decreased over the course of osteoblast differentiation. Overexpression of miR-206 in osteoblasts inhibited their differentiation, and conversely, knockdown of miR-206 expression promoted osteoblast differentiation. In silico analysis and molecular experiments revealed connexin 43 (Cx43), a major gap junction protein in osteoblasts, as a target of miR-206, and restoration of Cx43 expression in miR-206-expressing osteoblasts rescued them from the inhibitory effect of miR-206 on osteoblast differentiation. However, the regulating network formed by miRNA - 206 and Cx43 had not been explored in SANFH. We hypothesize the Cx43/ miRNA-206 through the regulation of MAPK/ERK 、PKC and Canonical Wnt signaling pathway inhibit the differentiation of osteoblast, which plays an important role in SANFH. In order to validate the effects of miRNA- 206 and Cx43 on SANFH to provide new targets, we are going to detecte the miRNA-206 and its target genes in patients with SANFH and SANFH model of rabbits by real-time quantitative PCR and the in situ hybridization. And transfect the rabbit head with overexpression and low expression of miRNA-206 viral vector, then detect the relevant indicators, to further confirm the role of miRNA-206 and its regulation of Cx43 in SANFH.

激素性股骨头坏死（SANFH）发病机制至今未明，成骨分化障碍与其密切相关。miRNA-206下调靶蛋白缝隙连接蛋白(Cx)43抑制成骨细胞分化，Cx43高表达可激活MAPK/ERK、PKC和Wnt/β-catenin信号通路，促进成骨细胞分化。Cx43/miRNA-206调控的成骨细胞分化在SANFH中的作用国内外均未见报道。我们假设Cx43/miRNA-206通过下游MAPK/ERK、PKC和Wnt/β-catenin信号通路，调控成骨细胞分化在SANFH中发挥重要作用。采用实时定量PCR、原位杂交等技术检测SANFH患者和SANFH兔股骨头miRNA-206、Cx43、ALP、ERK1/2、PKC、β-catenin等的表达，建立miRNA-206过表达/抑制表达SANFH兔模型，阐明Cx43/miRNA-206调控的成骨分化在SANFH中的作用及机制，为治疗SANFH提供新靶点。

背景：激素性股骨头坏死（steroid-induced avascularnecrosis of the femoral head，SANFH）发病机制至今未明，成骨分化障碍与其密切相关。前期研究表明，微小RNA-206（microRNA-206，miR-206）可下调其靶蛋白缝隙连接蛋白 43(connexin43，Cx43)继而抑制成骨细胞分化，Cx43 高表达可激活MAPK/ERK和Wnt/β -catenin 信号通路，促进成骨细胞分化。而 miR-206 靶向 Cx43 调控成骨分化在 SANFH 中的作用尚未见报道。.研究内容：本课题以激素性股骨头坏死患者及兔为研究对象，从临床和动物实验两方面，应用荧光定量PCR及Western blot法检测坏死股骨头组织成骨标志性基因碱性磷酸酶（ALP）、runt相关转录因子2（Runx2）表达水平，评估成骨分化能力；通过锁定的核苷酸原位杂交技术、荧光定量PCR、Western blot等方法检测miRNA-206及其靶蛋白Cx43、下游MAPK/ERK及经典的Wnt信号通路的表达变化。构建miRNA-206 抑制/过表达腺相关病毒载体，转染兔股骨头，动态观察成骨分化能力指标的变化。.结果：应用内毒素联合甲强龙可成功建立兔激素性股骨头坏死模型。成骨分化障碍与激素性股骨头坏死密切相关。在激素性股骨头坏死骨组织中，miR-206表达上调，Cx43表达下调。miR-206通过抑制Cx43的表达，从而抑制成骨细胞增殖、分化；miR-206通过下调其靶蛋白Cx43，抑制其下游MAPK/ERK及Wnt/β-catenin信号通路和转导，进而抑制成骨分化参与激素性股骨头坏死发生发展进程。.意义：本研究证实明miRNA-206及其调控的靶蛋白Cx43通过抑制成骨细胞分化在激素性股骨头坏死疾病中发挥重要作用，为股骨头坏死提供了一种新的分子标志物，为临床治疗提供新的理论依据和治疗靶点。