阿胶强骨口服液含药血清对胎鼠成骨细胞护骨素及护骨素配体mRNA表达的影响

Effects of containing drugs blood serum of donkey - hide glue reinforcing bone oral solution on mRNA expressing of OPG and OPGL in osteoblast in rats

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 investigate the effects of containing drugs blood serum of donkey - hide glue reinforcing bone oral solution (DGRBOS) on mRNA expression of OPG and OPGL in osteoblast in rats and explore the molecular mechanism of treating osteoporosis with DGRBOS. Methods 30 Wistar rats were randomly divided into DGRBOS group, estrogen group and normal control group. After intragastric administration for 7 days, containing drugs serum was prepared, Skull osteoblast was isolated from newborn SD rats and was made into single cell suspension. The cultured osteoblasts were divided into 5 groups. The experimental groups were given equal volumes of drugs serum and the control group was given cultured fluid. The osteoblast proliferation was measured by antigenic MTT colorimetric analysis and 3 H - TdR penetration method. The intra - cellular BGP contents were evaluated by radioimmunity. The mRNA expression of OPG and PANKL in osteoblasts was analyzed by Rt -PCR. Results DGRBOS could enhance osteoblast proliferation in a dose - dependent manner, compared to the control group; a significant difference was present (P<0. 05). As the concentration of contained drug serum of DGRBOS for 1000ml/L appeared as the peak of mRNA expression OPG and OPGL in osteoblasts, compared with control group, significant differences were absent (P>0. 05). Conclusion The partial mechanism of preventing and treating osteoporosis with DGRBOS is that it might promote osseous anabolism and preosteoblast caryocinesia at cellular lever and regulate OPG/ RANKL ratio at molecular lever.