microRNA在祛痰逐瘀法促酒精性股骨头坏死骨新生中的调控机制研究

Alcohol enhanced ability to stimulate BMSCs adipogenic, osteogenic diminished capacity is an important cause of osteonecrosis (ONFH) in patients with disorders of fat metabolism. Endogenous microRNA (miRNA) in BMSCs into adipocytes - from regulation of osteogenic differentiation. Chinese medicine believes that "phlegm" and "stasis" is closely related to lipid metabolism. Studies have shown that "Phlegm" may improve lipid metabolism in patients with alcoholic ONFH abnormal state, and can promote osteogenic differentiation of rabbit BMSCs. We speculate that there may be alcohol ONFH patient-specific miRNA involved in bone repair, and may be expectorant action Stasis target miRNA. This paper intends to use after treatment ① high-throughput screening assay ONFH alcoholic patients and expectorant stasis law changes in plasma miRNA expression profiling, screening differences in miRNA, QPCR validated miRNA target gene expression and predict; ② with miRNA mimetic or inhibitors in patients with BMSCs transfected ONFH differences of miRNA function and determine its target genes, transcriptional regulation of research expectorant stasis method of miRNA targets; ③ joint research expectorant stasis party most differentially expressed miRNA regulation of target gene transfection repair of rabbit BMSCs transplantation in alcoholic ONFH.

酒精刺激BMSCs成脂能力增强、成骨能力减弱是导致股骨头坏死（ONFH）患者脂肪代谢紊乱的重要原因。内源microRNA（miRNA）在BMSCs成脂-成骨分化中起调控作用。中医学认为，“痰”和“瘀”与脂代谢紊乱密切相关。研究表明“痰瘀同治”可改善酒精性ONFH患者脂代谢异常状态，且可促兔BMSCs成骨分化。我们推测酒精性ONFH患者可能存在特异miRNA参与骨修复，且可能是祛痰逐瘀法作用的靶miRNA。本课题①拟采用高通量筛选法检测酒精性ONFH患者及祛痰逐瘀法治疗后血浆中miRNA表达谱变化，筛选差异miRNA，QPCR验证miRNA表达并预测其靶基因；②用miRNA模拟剂或抑制剂转染ONFH患者BMSCs研究差异miRNA的功能并确定其靶基因，研究祛痰逐瘀法对靶miRNA的转录调控作用；③研究祛痰逐瘀方联合最差异表达miRNA调控的靶基因转染BMSCs移植修复兔酒精性ONFH的效果。

本项目共三个部分：①检测酒精性ONFH患者及祛痰逐瘀法(QZD)治疗后血浆中miRNA表达谱变化并预测其靶基因。分析入组的AIONFH患者的血清样品中miRNA的光谱，并应用随机方差模型（RVM）来过滤差异表达的miRNA，发现7种miRNA（包括miR-127-3p，miR-1，miR-654-3p，miR-628-3p，miR-432-5p，miR-582-3p和miR-323a-3p）显着下调，3种miRNA（miR-483-5p，miR-483-3p和miR-885-5p）显着上调；使用GO数据库和KEGG途径分析探索这些miRNA的功能和潜在的信号传导途径，结果发现所选miRNA能上调信号转导，细胞分化，细胞甲基化，细胞生长和凋亡等相关途径，此外与Wnt信号传导途径、PI3K-Akt和癌症途径的相关性最高。②研究差异表达miRNA的功能并确定其靶基因，继而研究QZD对靶基因的转录调控作用；在动物和人的实验中，都发现健康人和QZD治疗后的患者体内miＲ－628 表达量高于未经QZD治疗的AIONFH患者；运用RT-qPCR对动物模型骨组织进行检测，结果发现QZD可调控骨组织内Pten和Runx2基因表达，所以目前认为QZD能通过miR-628/Pten/Runx2通路，从而恢复AIONFH骨代谢稳态。③观察QZD对新生血管形成的干预。血管内皮细胞的聚集和组合是构成血管的基础，周细胞填补血管内皮细胞之间的空隙，使血管趋于稳定，此二者协同完成血管的重建，而血管重建对于包括骨组织在内的需要进行修复的组织器官非常重要。加入QZD对SVEC细胞进行干预后可显着增强了SVEC细胞迁移，说明ＱＺＤ含药血清对于血管内皮细胞的分化具有积极的作用，免疫荧光染色实验也证实QZD可促进周细胞的聚集。由此说明ＱZＤ对于血管重建的作用具有多方面、立体化、复合性的特点，能激活破骨细胞和成骨细胞，促进骨吸收和骨形成，从而维持骨稳态平衡。