补肾壮骨中药对成骨细胞OPG、RANKL mRNA表达的影响

The effect of traditional Chinese medicine on the expression of mRNA of OPG and RANKL

目的观察补肾壮骨中药对成骨细胞OPG和RANKL的影响,探讨中药对牵张成骨作用机制。方法 Beagle犬6只,雄性,体重10kg^15kg。实验分为含药血清组(A组)、无药血清组(B组)和胎牛血清组(C组)。含药血清组的血清来自补肾壮骨中药经煎制后,按体表面积折算等效剂量,2只Beagle犬连续喂服7天后经股动脉采血制备而成。无药血清组的血清采用等剂量生理盐水2只Beagle犬喂服7天后经股动脉采血制备而成。胎牛血清组的血清直接购买。另外2只Beagle犬由胫骨获取骨髓,经F icoll分离液进行梯度离心,MSC经含胎牛血清的DMEM培养,传代后分别将MSC在含药血清(A组)、无药血清(B组)和胎牛血清(C组)的诱导矿化液(β-甘油磷酸钠,维生素C和地塞米松)+DMEM中培养。采用原位杂交的方法检测成骨细胞OPG和RANKL mRNA的表达情况。结果诱导培养5天、7天时,含药血清组成骨细胞OPG mRNA的表达量均高于无药血清组和胎牛血清组(P<0.05)。无药血清组和胎牛血清组相比无显著性差异(P>0.05)。诱导培养5天时,成骨细胞RANKLmRNA的表达量三组间两两比较均无显著性差异(P>0.05)。诱导培养7天时,含药血清组成骨细胞OPG mRNA的表达量均低于无药血清组和胎牛血清组(P<0.05)。无药血清组和胎牛血清组相比无显著性差异(P>0.05)。诱导培养5天时,含药血清组OPG/RANKL比率是无药血清组的1.67倍,是胎牛血清组的2.01倍。诱导培养7天时,含药血清组OPG/RANKL比率是无药血清组的2.18倍,是胎牛血清组的2.62倍。结论补肾壮骨中药可以调节成骨细胞分泌OPG、RANKL的功能,从而降低破骨细胞的活力,促进成骨过程。

Objective To observe the effect of traditional Chinese medicine on the expression of OPG and RANKL and to investigate the mechanism of distracting osteogenesis.Methods Six male Beagle dogs,each weighed 10kg～15kg,were divided into three groups： group A medicine serum group,group B non-medicine serum group and group C bovine serum group respectively.The serum of group A was obtained from the femoral artery of 2 Beagle dogs that had been given equivalent dose of traditional Chinese medicine for 7 days continuously according to body surface area.The serum of group B was also collected from the femoral artery of 2 Beagle dogs fed with equal volume of normal saline water for 7 days continuously.The serum of group C was bought fetal bovine serum.The tibia marrow was harvested from another 2 Beagle dogs and MSCs were isolated and purified by density gradient centrifugation.Then MSCs were cultured in DMEM solution with fetal bovine serum.MSCs of the 3rd generation were cultured in DMED medium with mineralization inducing liquid containing medicine serum（group A）,non-medicine serum（group B） and bovine serum（group C） respectively.The expression of osteoblast OPG and RANKL mRNA were detected by situ hybridization method.Results After induced culturing for 5 days and 7 days,the expression levels of osteoblasts OPG mRNA of medicine serum group was higher than that of non-medicine serum group and bovine serum group（P0.05）.But non-medicine serum group and bovine serum group showed no significant difference（P0.05）.After induced culturing for 5 days,the expression levels of osteoblasts RANKL mRNA of the three groups had no significant differences（P0.05）.After induced culturing for 7 days,the expression levels of osteoblasts RANKL mRNA of medicine serum group were less than that of non-medicine serum and bovine serum（P0.05）.But non-medicine serum group and bovine serum group showed no significant difference（P0.05）.After induced culturing for 5 days,OPG/RANKL ratio of medicine serum group was 1.67 compared with non-medicated serum group and 2.01 compared with bovine serum group.After induced culturing for 7 days,OPG / RANKL ratio of medicine serum was 2.18 compared with non-medicine group and 2.62 compared with bovine serum group.Conclusion The traditional Chinese medicine can promote the synthesis of osteoblast OPG,inhibit the release of osteoblast RANKL,and increase OPG/ RANKL ratio,then reduce the vitality of osteoclasts and promote the forming of new bone.