OTX2基因对hFOB1.19成骨细胞增殖和分化的影响

Effect of OTX2 gene on the proliferation and differentiation of hFOB1.19 osteoblasts

背景:研究成骨细胞的表达及分化有助于阐明骨性反牙合畸形下颌骨发育过度的分子机制。目的:应用RNA干扰技术下调成骨细胞系hFOB1.19中OTX2基因的表达,进一步研究OTX2基因对成骨细胞增殖及分化的影响。方法:实验分为6组:特异性OTX2-siRNA1、OTX2-siRNA2、OTX2-siRNA3组,GAPDH-siRNA组,阴性对照组和空白对照组。设计3个靶序列的小干扰RNA(iRNA),转染人成骨细胞hFOB 1.19。RT-PCR检测转染后成骨细胞中OTX2 mRNA水平的变化;Western Blot检测蛋白质表达水平;MTT法评估OTX2-si RNA对成骨细胞增殖的抑制作用;化学比色法检测细胞碱性磷酸酶活性。结果与结论:(1)转染后,出现OTX2 mRNA水平下调,蛋白表达水平下调;(2)倒置显微镜下观察漂浮细胞OTX2-si RNA组明显多于各对照组,细胞增殖抑制率明显高于各对照组,OTX2-si RNA组碱性磷酸酶活性明显降低;(3)结果提示,化学合成的特异性OTX2-siRNA转染人成骨细胞系hFOB1.19,能有效下调OTX2mRNA水平;OTX2基因表达下调能够抑制hFOB1.19成骨细胞增殖;OTX2基因表达下调可降低h FOB1.19成骨细胞碱性磷酸酶活性,抑制其分化。

BACKGROUND： Studying the expression and differentiation of osteoblasts contributes to understanding the mechanism of hereditary mandibular deformity at molecular level. OBJECTIVE： To down-regulate the expression of OTX2 gene and investigate the effect of OTX2 gene on the proliferation and differentiation of hFOB1.19 osteoblasts by RNA interference. METHODS： There were six groups： synthesized OTX2-siRNA1, OTX2-siRNA2, OTX2-siRNA3, GAPDH-siRNA, negative control, and blank control groups. Three target sequence si RNAs were designed and transfected intothe osteoblasts. The OTX2 mRNA level in osteoblasts was detected by RT-PCR after transfection. The protein expression level was detected by western blot assay. The inhibition of OTX2-siRNA on osteoblast proliferation was evaluated by MTT assay. The alkaline phosphatase activity was detected by chemical colorimetry. RESULTS AND CONCLUSION： OTX2 m RNA and protein expression was down-regulated after transfection. In the OTX2-si RNA group, the number of loating cells and cell proliferation inhibition rate were higher and the alkaline phosphatase activity was decreased compared with the control group. Our findings indicate that the chemically synthesized OTX2-si RNA can effectively restrain m RNA expression level of OTX2 gene in hFOB1.19 osteoblasts. Interference of OTX2 gene can inhibit the proliferation of hFOB1.19 osteoblasts, and down-regulated expression level of OTX2 gene can reduce the alkaline phosphatase activity of hFOB1.19 osteoblasts and inhibit their differentiation.