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目的观察重组结核杆菌热休克蛋白10(CPN10)对成骨细胞(OB)-外周血单个核细胞(PBMs)共培养体系中破骨细胞生成及相关基因表达的影响。方法建立培养上清相通但二者互相不接触的成骨细胞一单个核细胞共育模型。实验分对照组和CPN10(10μg/ml)处理组。主要观察指标:1采用TRAP染色及扫描电镜检测破骨细胞生成及小牛骨磨片吸收陷窝,2应用Realtime PCR检测与破骨细胞生成相关基因NFATc1、c-Fos、RANKL、OPG的基因表达。结果两组细胞均有TRAP阳性多核破骨细胞生成,并在小牛骨磨片上形成吸收陷窝;但对照组所获TRAP阳性多核细胞数目、吸收陷窝数目及面积均显著小于CPN10组。Realtime PCR检测结果显示CPN10组与对照组相比NFATc1、c-Fos、RANKL、OPG相对浓度分别为33.4798±2.0929、47.974±5.1628、47.0861±2.2033、7.4642±0.6791(P<0.05),对照组各基因表达均显著低于CPN10组。结论 CPN10在成骨细胞-单个核细胞(OB-PBMs)共培养体系中可促进OC的生成及骨吸收,CPN10通过对成骨细胞的作用,致其分泌的OPG/RANKL比例失调,并上调破骨细胞相关基因NFATc1、c-Fos、RANKL、OPG的基因表达。
Objective To investigate the effect of recombinant Mycobacterium tuberculosis heat shock protein 10 (CPN10) on osteoclastogenesis and related gene expression in osteoblasts (OB) -peripheral blood mononuclear cells (PBMs) co-culture system. Methods The osteoblast-mononuclear cell co-culture model was established to establish a culture supernatant but not to contact with each other. Experimental control group and CPN10 (10μg / ml) treatment group. MAIN OUTCOME MEASURES: 1 TRAP staining and scanning electron microscopy were used to detect osteoclastogenesis and calf bone resorption lacuna. 2 Realtime PCR was used to detect the gene expression of osteoclastogenesis-related genes NFATc1, c-Fos, RANKL and OPG . Results TRAP-positive multinucleated osteoclasts were formed in both groups, and lacunae formed on the calf disc. However, the number of TRAP-positive multinucleated cells and the number of lacunae in the control group were significantly smaller than those in CPN10 group. Realtime PCR results showed that the relative concentrations of NFATc1, c-Fos, RANKL and OPG in CPN10 group were 33.4798 ± 2.0929,47.974 ± 5.1628,47.0861 ± 2.2033 and 7.4642 ± 0.6791, respectively (P <0.05) The expression was significantly lower than CPN10 group. CONCLUSION: CPN10 promotes OC production and bone resorption in osteoblasts and mononuclear cells (OB-PBMs) co-culture system. The effect of CPN10 on osteoblasts results in imbalanced OPG / RANKL secretion and up-regulation Gene expression of osteoclast related genes NFATc1, c-Fos, RANKL and OPG.