RNA干涉抑制破骨细胞分化因子表达对骨髓基质细胞成骨成脂分化的影响

背景：RNA干涉是目前分子生物学研究领域重要的基因下调技术,护骨素/κB受体活化因子/破骨细胞分化因子偶联系统是目前骨改建平衡研究中的热点。目的：应用RNA干涉特异性抑制大鼠骨髓基质细胞内破骨细胞分化因子基因的表达,研究破骨细胞分化因子表达下调后对骨髓基质细胞成骨成脂分化的影响。方法：获取骨髓基质细胞,转染前24h按5×105/孔接种于6孔培养板中,分为实验组、阴性对照组和空白对照组,前2组细胞分别转染针对破骨细胞分化因子的siRNA或阴性对照siRNA。观察骨髓基质细胞增殖活性、碱性磷酸酶活性及Real-TimePCR检测成骨成脂基因mRNA的表达。结果与结论：实验组碱性磷酸酶活性降低,RunX-2,骨形态发生蛋白2,4表达降低,而PPAR-γ和C/EBP-α的表达升高（P〈0.05）。提示,通过RNA干涉使骨髓基质细胞的破骨细胞分化因子表达下调对骨髓基质细胞成骨分化有抑制作用,而对成脂分化有促进作用。

Effect of receptor activator of nuclear factor kappa B ligand expression suppressed by RNA interference on osteogenic and adipogenic differentiation potential of bone marrow derived stroma cells

BACKGROUND：RNA interference（RNAi） is an important method to down-regulate the gene expression in molecular biological study. Osteoprotegerin（OPG） /receptor activator of nuclear factor kappa B（RANK） /RANK ligand（RANKL） couple system is a hotspot in the research of bone remolding. OBJECTIVE：To investigate the effect of RANKL expression suppressed by RNAi on osteogenic and adipogenic differentiation potential of bone marrow derived stroma cells（BMSCs） . METHODS：BMSCs were isolated and cultured from SD rats,then were seeded in 6-holes culture plates by 5 × 105 /hole 24 hours before transfection and divided into experimental group,negative control group and blank group. siRNA was transfected into the cells by Lipofectin 2000TM in experimental group and negative control group. Proliferation activity of BMSCs and alkaline phosphatase activity were measured,and the mRNA expression revolving in osteogenic and adipogenic differentiation of BMSCs were detected by real-time PCR. RESULTS AND CONCLUSION：Alkaline phosphatase activity,RunX-2,bone morphogenetic protein-2（BMP-2） ,and BMP-4 expression decreased,while peroxisome proliferator-activated receptor-γ and recombinant Human CCAAT/enhancer binding protein alpha expression increased after RANKL expression inhibited by RNAi（P 0.05） . The results suggest that the down-regulation of RANKL by RNAi could improve adipogenic differentiation and depress osteogenic differentiation of BMSCs.