补肾舒脊颗粒含药血清对人骨髓间充质干细胞Wnt5a、β-catenin mRNA及蛋白表达的影响

Effect of Bushen Shuji Granules(补肾舒脊颗粒) Drug Containing Serum on mRNA and Protein Expressions of Wnt5a and β-catenin in Human Bone Marrow Mesenchymal Stem Cells

目的探讨补肾舒脊颗粒延缓异位骨化的可能作用机制。方法体外培养人骨髓间充质干细胞(BMSCs)分为空白组、模型组、对照组及中药高、中、低剂量组。空白组加入基础培养基(3.8 ml)+5%正常大鼠血清(0.2 ml);模型组加入诱导培养基(4.8 ml)+5%正常大鼠血清(0.2 ml);西药组加入诱导培养基(3.8 ml)+5%洛索洛芬钠含药血清(0.2 ml);中药高、中、低剂量组分别加入诱导培养基(3.8 ml)+5%补肾舒脊颗粒含药血清(0.2 ml)。28天后通过逆转录-聚合酶链反应(RT-PCR)和免疫印迹试验(Western blot)检测Wnt5a mRNA、β-catenin mRNA及蛋白表达。结果与空白组比较,模型组Wnt5a、β-catenin蛋白及mRNA表达均升高(P<0.05或P<0.01);与模型组比较,对照组和中药各剂量组Wnt5a、β-catenin蛋白及mRNA表达均降低(P<0.05或P<0.01);与对照组比较,中药高剂量组Wnt5a mRNA、β-catenin mRNA表达降低,中药中剂量组Wnt5a、β-catenin蛋白表达降低(P<0.05或P<0.01);与中药低、中剂量组比较,中药高剂量组Wnt5a mRNA、β-catenin mRNA表达降低(P<0.05或P<0.01)。与中药低剂量组比较,中药中剂量组Wnt5a蛋白表达降低,中药高剂量组β-catenin蛋白表达升高(P<0.05)。结论补肾舒脊颗粒可能通过Wnt/β-catenin信号通路抑制Wnt5a mRNA、β-catenin mRNA及蛋白的表达,从而起到延缓强直性脊柱炎异位骨化的作用。

Objective To explore the possible mechanism of Bushen Shuji Granules（补肾舒脊颗粒,BSG） in delaying heterotopic ossification. Methods Human bone marrow mesenchymal stem cells（BMSCs） were cultured in vitro and divided into blank group,model group,control group and Chinese medicine high,medium and low dose groups. The blank group was cultured with basal medium（3. 8 ml） ＋ 5% normal rat serum（0. 2 ml）; the model group was cultured with induction medium（4. 8 ml） ＋ 5% normal rat serum（0. 2 ml）; the control group was cultured with induction medium（3. 8 ml） ＋ 5% loxoprofen sodium containing serum（0. 2 ml）; the Chinese medicine high,medium and low dose groups were cultured with induction medium（3. 8 ml） ＋ 5% BSGs containing serum（0. 2 ml）. After 28 days,the mRNA and protein expressions of Wnt5 a and β-catenin were detected by reverse transcription-polymerase chain reaction（RT-PCR） and Western blotting. Results Compared with those in the blank group,Wnt5 a and β-catenin protein and mRNA expressions in the model group significantly increased（P 〈0. 05 or P 〈0. 01）. Compared with those in the model group,the mRNA and protein expressions of Wnt5 a and β-catenin in the control group and Chinese medicine high,medium and low dose groups decreased（P 〈0. 05 or P 〈0. 01）; compared with those in the control group,the mRNA expressions of Wnt5 a and β-catenin in Chinese medicine high dose group decreased,while the protein expressions of Wnt5 a and β-catenin in Chinese medicine medium dose group decreased（P 〈0. 05 or P 〈0. 01）; compared with those in Chinese medicine low and medium dose groups,the mRNA expressions of Wnt5 a and β-catenin in the high dose group decreased（P 〈0. 05 or P 〈0. 01）. Compared with those in Chinese medicine low dose group,the Wnt5 a protein expression in the medium-dose group decreased,and the β-catenin protein expression in the high-dose group increased（P 〈0. 05）. Conclusion BSG may inhibit the mRNA and protein expressions of Wnt5 a and β-catenin through the Wnt/β-catenin signaling pathway,which can delay the heterotopic ossification of ankylosing spondylitis.