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目的:探讨smad3是否介导了补肾中药对增龄成骨细胞VDR mRNA表达的影响,从而阐明补肾中药具有促进骨形成的机制。方法:分离6月龄和16月龄SD大鼠成骨细胞,采用组织块翻转方法培养,通过倒置显微镜观察细胞形态,并以矿化结节染色对成骨细胞加以鉴定。将SD大鼠随机分为正常组、生理盐水组、单味补阳组(固本壮骨粉)、复方补阳组(金匮肾气丸)、复方平补组(补肾益精方)、复方补阴组(知柏地黄丸)和西药对照组(阿法骨化醇片),制备药物血清;当成骨细胞汇合达60%,换无血清培养液培养细胞24 h后,加入含药血清(浓度为10%)再继续培养3 d。采用RT-PCR方法检测含药血清干预后成骨细胞smad3和VDR mRNA的表达。结果:在增龄成骨细胞smad3 mRNA表达是下降的,这与增龄所致VDR mRNA表达下降相一致;单味补阳组、复方补阳组和西药对照组上调smad3 mRNA的趋势与它们上调VDR mRNA的趋势相一致。结论:补阳中药可能通过启动Smad3 mRNA表达而上调VDR mRNA表达,从而促进骨形成。
Objective: To investigate whether smad3 mediates the effect of kidney-reinforcing traditional Chinese medicine on the expression of VDR mRNA in aging osteoblasts, thus clarifying the mechanism by which tonifying kidney herbs can promote bone formation. METHODS: Osteoblasts were isolated from 6-month-old and 16-month-old SD rats and cultured using a tissue block inversion method. Cell morphology was observed by inverted microscope and osteoblasts were identified by staining with mineralized nodules. SD rats were randomly divided into normal group, normal saline group, single-tonic tonic group (Guben Zhuanggu powder), compound Bu Yang group (Jinkuishenqi Pills), compound Pingbu group (Bu Shen Yi Jing Fang), and compound prescriptions. Yin Yin group (Zhibai Dihuang Pills) and Western medicine control group (Alfacalcitol tablets), preparation of drug serum; when the osteoblasts confluence of 60%, for 24 hours after the culture medium without serum, cultured serum was added ( Concentration is 10%) continue to culture for 3 days. The expression of smad3 and VDR mRNA in osteoblasts was detected by RT-PCR. Results: The expression of smad3 mRNA in aged osteoblasts was decreased, which was consistent with the decrease of VDR mRNA expression induced by aging; the trend of up-regulating smad3 mRNA and their up-regulation in single-tonic tonic group, compound yang group and western medicine control group. The trend of VDR mRNA is consistent. Conclusion: Chinese herbal medicine for tonifying yang may up-regulate the expression of VDR mRNA by initiating the expression of Smad3 mRNA and promote bone formation.