结核分枝杆菌裂解物诱导破骨细胞及OPG／RANKL表达变化实验研究

摘要

目的探讨结核分枝杆菌超声裂解产物（Mt sonicate）体外诱导小鼠破骨细胞的形成及 OPG/RANKL表达变化与裂解物的浓度、时间的依赖性，研究结核杆菌引起骨破坏的相关机制。方法原代培养的 BALB/c小鼠骨髓基质细胞以高、中、低浓度的 Mt sonicate进行干预，在1、3、7、14 d进行 TRAP染色，观察破骨细胞的形成，并采用 Realtime RT-PCR检测各浓度、时间的骨保护素（OPG）/核因子Kappa B受体活化因子配基（RANKL）mR-NA表达。结果 Mt sonicate在体外可诱导小鼠骨髓基质细胞向破骨细胞的转化；破骨细胞数量与 Mt sonicate浓度在3、7 d时呈显著的正相关（3 d：r＝0．417，P ＜0．05；7 d：r＝0．463，P ＜0．05）；3个浓度干预下，破骨细胞数量在1～7 d逐渐增多，7～14 d则逐渐减少。7 d时，高、中、低浓度 Mt sonicate干预下 OPG/RANKL mRNA表达比分别为（1．35±0．11）、（7．38±1．15）、（9．85±1．26）。结论小鼠骨髓基质细胞向破骨细胞转化的实验中，存在结核分枝杆菌裂解物的浓度和时间依赖性，且与 OPG/RANKL mRNA的表达比呈负相关，破骨细胞形成时的数量与活性受到结核分枝杆菌的调控，其分子机制与 OPG/RANKL系统的变化密切相关。%Objective This paper aims to explore the formation of broken bone cell in vitro mouse bone marrow stromal cells by Mycobacterium tuberculosis ultrasound pyrolysis products (Mt sonicate ) induction,the lysate concentration dependence of the time,and the mechanisms of bone destruction caused by TB bacilli.Methods Primary cultures of BALB/c mice bone marrow stromal cells were intervened with high,medium and low concentrations of Mt sonicate on 1,3,7,14 d;osteoclast formation was observed with TRAP staining and Realtime RT-PCR to detect the concentration and time of the OPG/RANKL mR-NA expression.Results Mt sonicate can induce in vitro transformation of mouse bone marrow stromal cells into osteoclasts;the number of osteoclasts and Mt sonicate concentration on 3 d and 7 d showed a sig-nificant positive correlation(3 d:r=0.417 P <0.05;7 d:r=0.463 P <0.05);with three concentrations intervention,the number of osteoclasts gradually increased from 1 d to 7 d,gradually reduced from 7 to 14 d;on 7 d,OPG/RANKL mRNA expression ratios in each group of Mt sonicate intervened cells in high medium and low concentrations were 1.35 ± 0.11,7.38 ± 1.15,9.85 ± 1.26.Conclusion There exist the dependence of Mycobacterium tuberculosis lysate concentration and time in the experiments on mouse bone marrow stromal cells into osteoclasts transformation,and the ratio of OPG/RANKL mRNA expression showed a negative relevance;the quantity and activity of osteoclastogenesis was regulated by the Mycobac-terium tuberculosis;the molecular mechanisms of changes are closely related with OPG/RANKL system.