目的 研究天山雪莲愈伤组织提取物(extract from callus of Saussurea involucrate, ESI)对人成骨肉瘤细胞MG-63增殖及分化的影响,探讨ESI抗骨质疏松的作用及机制。方法 以MG-63为研究对象,采用MTT法和乳酸脱氢酶(LDH)法检测ESI对成骨细胞增殖的影响,相关试剂盒检测碱性磷酸酶(ALP)活性和羟脯氨酸(Hyp)水平,茜素红染色法观察对矿化骨结节形成的影响;实时荧光定量PCR(RT-PCR)检测骨保护素(OPG)和核因子-κB活化因子受体配体(RANKL)基因表达水平;Western blotting法检测OPG/RANKL蛋白水平;检测p38特异性抑制剂SB203580对ESI作用于成骨细胞增殖、分化、矿化以及OPG/RANKL表达的影响。结果 ESI低于125μg/mL对成骨细胞无细胞毒性,31.25、62.5μg/mL对成骨细胞生长有促进作用;ESI显著提高ALP活性、Hyp水平以及矿化骨结节形成;显著上调MG-63细胞OPG mRNA水平,下调RANKL mRNA水平,上调OPG/RANKL蛋白水平;SB203580显著抑制ESI对成骨细胞增殖、ALP活性、矿化结节形成的促进作用以及对胶原蛋白积累量和OPG/RANKL值的提高作用。结论 ESI能够促进成骨细胞增殖、分化和矿化,其机制可能与调节OPG和RANKL基因表达有关,并且可能通过p38丝裂原活化蛋白激酶(MAPK)通路进行信号转导。

Objective To investigate the effect of extract from callus of Saussurea involucrate (ESI) on the proliferation and differentiation of osteosarcoma cells MG-63 and explore its anti-osteoporosis and mechanism. Methods Using human osteosarcoma cells MG-63 for the study, the effect of ESI on the proliferation was detected by MTT and LDH methods; The activity of alkaline phosphatase activity (ALP) and the level of hydroxyproline (Hyp) were investigated by related reagent kit; The effect of ESI on mineralized nodules was observed by Alizarin red staining and quantified by CPC, the expression of OPG/RANKL was measured by RT-PCR and Western blotting, and the proliferation, differentiation, mineralization, and OPG/RANKL expression levels of MG-63 were assayed when medium contains both p38 inhibitor SB203580 and ESI. Results MTT and LDH assay showed that ESI (< 125 μg/mL) was non-toxic to MG-63, and it promoted the proliferation at the concentration of 31.25 and 62.5 μg/mL; ESI could significantly increase the ALP activity, Hyp content, and amount of mineralized nodules; At the mRNA level, ESI could significantly up-regulate OPG expression and down-regulate RANKL expression; At the protein level, ESI could significantly increase the ratio of OPG/RANKL; SB203580 could reverse the acceleration of ESI on the proliferation, differentiation, mineralization, and OPG/RANKL expression of MG-63. Conclusion ESI has the facilitating effect on the osteoblast proliferation, differentiation, and mineralization; The mechanism may be associated with the expression of OPG and RANKL, as well as the signal transduction pathway of p38 Mitogen-activated potein kinase (MAPK).