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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 effects of Bushen Zhuanggu Chinese herbs on OPG and RANKL of osteoblasts and explore the mechanism of traditional Chinese medicine on distraction osteogenesis. Methods Beagle dogs 6, male, weighing 10kg ~ 15kg. The experiment was divided into drug-containing serum group (A group), drug-free serum group (B group) and fetal bovine serum group (C group). Serum containing serum from the Bushen Zhuanggu Chinese medicine after frying, according to the body surface area equivalent dose, two Beagle dogs were fed for 7 days after the femoral artery blood preparation. The blood serum of the non-drug serum group was prepared by blood sampling in the femoral artery after 2 doses of normal saline and 2 Beagle dogs were fed for 7 days. Fetal bovine serum group purchased directly. The other two Beagle dogs were harvested from the tibia bone marrow by Ficoll separation fluid gradient centrifugation, MSCs were cultured in fetal bovine serum-containing DMEM after passage, the MSC in the drug-containing serum (A group), no drug serum (B group) And fetal calf serum (group C) in the induction mineralized solution (β-glycerophosphate, vitamin C and dexamethasone) + DMEM. The expression of OPG and RANKL mRNA in osteoblasts was detected by in situ hybridization. Results After cultured for 5 days and 7 days, the expression of OPG mRNA in osteoblasts containing drug-containing serum was higher than those in non-drug serum and fetal calf serum (P <0.05). No drug-serum group and fetal bovine serum compared to no significant difference (P> 0.05). The expression of RANKL mRNA in osteoblasts showed no significant difference among the three groups (P> 0.05). The expression of OPG mRNA in osteoblasts containing drug-containing serum for 7 days was lower than that of drug-free serum and fetal bovine serum (P <0.05). No drug-serum group and fetal bovine serum compared to no significant difference (P> 0.05). The induced OPG / RANKL ratio in the drug-containing serum group was 1.67 times higher than that in the non-drug serum group and 2.01 times higher than that of the fetal calf serum group at 5 days. When cultured for 7 days, the ratio of OPG / RANKL in drug-containing serum group was 2.18 times that of drug-free serum group and 2.62-fold in fetal calf serum group. Conclusion Bushen Zhuanggu herbs can regulate the function of osteoblasts to secrete OPG and RANKL, so as to reduce the activity of osteoclasts and promote the process of osteogenesis.