[关键词]
[摘要]
目的 探讨梅花鹿茸Ι型胶原(SPC-Ι)对破骨细胞的影响及其分子机制。方法 采用全骨髓细胞诱导法培养破骨细胞、成骨细胞。设对照组(给予完全培养液)、诱导组(给予高糖-DMEM诱导液)、SPC-Ι(2.5、5、10 g/L)组,除对照组外,其他组给予含核因子-κB受体活化因子配体(RANKL)、巨噬细胞集落刺激因子(M-CSF)各40 ng/mL的高糖-DMEM诱导液,条件培养7 d,每隔3天更换培养液补足药物质量浓度。经HE染色、抗酒石酸盐性磷酸酶(TRAP)染色,倒置显微镜下观察细胞形态;分光光度计检测TRAP活性,RT-PCR法检测TRAP、核因子-κB受体活化因子(RANK)、RANKL、骨保护素(OPG)基因的表达,Western blotting法检测RANK蛋白的表达。结果 与破骨细胞组比较,SPC-Ι 5、10 g/L组TRAP阳性细胞减少,TRAP活性降低,TRAP、RANK、RANKL基因的表达及RANK蛋白的表达下调(P<0.01);与对照组比较,质量浓度2.5、10 g/L组OPG基因表达上调,RANKL/OPG的值减小(P<0.01),2.5 g/L质量浓度组的作用不显著。结论 SPC-Ι对破骨细胞的生成及分化具有抑制作用,该作用通过RANKL/OPG信号转导途径调控TRAP基因、RANK基因的表达实现的。
[Key word]
[Abstract]
Objective To explore the effect of sika pilose antler type I collagen (SPC-I) on osteoclast and its molecular mechanism. Methods The osteoclasts and osteoblasts were cultured by the induction method of whole bone marrow cells. The control (with full medium), osteoclasts (with HG-DMEM inducing medium), and SPC-I (2.5, 5, and 10 g/L) groups were set up. Except the control group, others were given the HG-DMEM inducing medium with each 40 ng/mL of both RANKL and macrophage colony-stimulating factor (M-CSF), then conditioned cultured for 7 d, every other 3 d to replace medium for the complement of the drug concentration. By HE and tartrate-resistant acid phosphatase (TRAP) stainings, the cell morphology was observed under inverted microscope. The TRAP activity was detected using spectrophotometer, the gene expression of TRAP, receptor activator of NF-κB (RANK), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG) was measured by RT-PCR, and the RANK protein expression was detected by Western blotting. Results Compared with the osteoclast group, SPC-I (5 and 10 g/L) groups could make TRAP positive cells and TRAP activity decreased, TRAP, RANK, and RANKL expression in gene level reduced, and RANK expression in protein level down-regulated also (P < 0.01); Compared with the control group, SPC-I (2.5 and 10 g/L) could make the OPG expression in gene level increased and the RANKL/OPG ratio declined (P < 0.01). The effect of 5 g/L SPC-I was the most significant (P < 0.01). The effect of 2.5g/L SPC-I was not significant. Conclusion SPC-I has the inhibitory effect on the osteoclast formation and differentiation; The effect of implementation is through RANKL/OPG signal transduction pathway to regulate the expression of TRAP and RANK genes.
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[基金项目]
国家自然科学基金资助项目(81073158)