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目的研究阿胶强骨口服液含药血清对胎鼠成骨细胞中OPG、RANKL表达的影响,探讨阿胶强骨口服液治疗骨质疏松的分子机制。方法 3月龄Wistar大鼠(雌雄各半)30只,随机分为阿胶强骨口服液,雌激素,及生理盐水对照组,灌胃7d后,制备实验组和对照组的含药血清。将新生SD大鼠分离的颅骨成骨细胞,制成单细胞悬液,消化传代,取二代培养的成骨细胞,制成细胞悬液。培养细胞被分为5组,分别加同体积药液,对照组仅加培养液,继续培养。采用MTT比色分析、3H-胸腺嘧啶核苷(3H-TdR)渗入法测定成骨细胞增殖,用放免法测定细胞内BGP含量,RT-PCR测定胎鼠成骨细胞中OPG、RANKL mRNA的表达。结果阿胶强骨口服液含药血清以剂量依赖方式促进成骨细胞的增殖,与对照组相比,差异显著(P<0．05)。成骨细胞OPG基因mRNA表达在阿胶强骨口服液含药血清100ml／L时最强,与雌激素组比较无显著性差异(P>0 05),且明显高于对照组(P<0．05)。成骨细胞RANKL基因mRNA表达在1000ml／L阿胶强骨口服液含药血清与雌激素组比较无显著性差异(P<0 05),且明显低于对照组(P<0．05)。结论阿胶强骨口服液可在细胞水平上促进骨的合成代谢及前成骨细胞的有丝分裂,分子水平上调节 OPG／RANKL表达而治疗骨质疏松。
Objective To study the effect of serum containing Ejiao Qiangu oral liquid on the expression of OPG and RANKL in fetal rat osteoblasts and to explore the molecular mechanism of Ejiao Qianggu oral solution in the treatment of osteoporosis. Methods Thirty Wistar rats (half male and female) aged 3 months were randomly divided into asaru gum strong oral liquid, estrogen and normal saline control group. After intragastric administration for 7 days, the drug-containing serum of experimental group and control group was prepared. Separated calvarial osteoblasts from neonatal Sprague-Dawley rats were used to prepare single cell suspensions, which were digested and passaged. Second-generation cultured osteoblasts were used to prepare cell suspensions. The cultured cells were divided into 5 groups and the same volume of drug solution was added respectively. In the control group, only culture medium was added and the culture was continued. Osteoblast proliferation was determined by MTT assay and 3H-TdR infiltration assay. BGP content in cells was determined by radioimmunoassay. OPG and RANKL mRNA expression in fetal rat osteoblasts was determined by RT-PCR. . Results The serum containing Ejiao Qiangu oral liquid promoted the proliferation of osteoblasts in a dose-dependent manner, which was significantly different from the control group (P<0.05). OPG gene mRNA expression in osteoblasts was the strongest in the serum containing 100 ml/L of Ejiao Qiangu oral solution, and there was no significant difference (P>0.05) from the estrogen group, which was significantly higher than that of the control group (P<0. 05). The mRNA expression of RANKL mRNA in osteoblasts was not significantly different from that in the estrogen group (P<0.05), and was significantly lower than that in the control group (P<0.05). Conclusion Ejiao Qianggu Oral Liquid can promote bone anabolism and mitosis of preosteoblasts at the cellular level, regulate OPG/RANKL expression at the molecular level, and treat osteoporosis.