共培养体系中重组结核杆菌热休克蛋白10对破骨细胞相关基因表达的影响

The influence of r-Mt cpn10 on the expression of osteoclast associated genes in an Osteoblast-Peripheral Blood Monocytes co-culture system

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 study the influence of the recombinant mycobacterium tuberculosis heat shock protein 10 （CPN10） on osteoclastogenesis, bone resorption and the expression of osteoclast-associated genes in an osteoblast （OB） -Peripheral Blood Monocytes （PBMs） co-culture system. Methods The osteoblast-monocyte co-culture system was estab-lished by developing the supernatant interaction system which could disable the contact of supernatants. The cells was co-cultureed with M-CSF （30 ng/ml）. CPN10 （10μg/ml） were added to form the CPN10 group, and the system without CPN10 was the control group. The primary parameters observed in this study were： ①morphology and growth of the osteo-clasts; ②Osteoclastogenesis examined by TRAP staining and their resorption lacunaes observed by scanning elec-tron microscope（SEM）;③Osteoclast formation related genes expression such as NFATc1, c-Fos, RANKL and OPG detected by Real-time PCR. Results TRAP positive multinuclear cells and bone resorption lacunaes were observed in both groups. However, CPN10 group showed more TRAP positive multinuclear cells and more resorption lacunaes than the control group. Real-time PCR detection results showed that NFATc1, c-Fos, RANKL and OPG gene expression of the control group were significantly lower than those of the CPN10 group （33.4798＆#177;2.0929、47.974＆#177;5.1628、47.0861＆#177;2.2033、7.4642＆#177; 0.6791, P〈0.05）. Conclusion CPN10 in OB and PBMs co-culture system can promote the formation of OC and bone resorption. CPN10 can make the secretion of OPG/RANKL ratio imbalance in osteoblasts, and stimulate the expression of OC related genes including NFATc1, c-Fos, RANKL and OPG.