梓醇对 OB-OC 共育体系中 OB、OC 活性及 OB ERα、βmRNA 表达的影响

摘要

目的：观察中药单体梓醇在成骨-破骨共育体系中对成骨细胞（OB）增殖、OB碱性磷酸酶（ALP）活性、破骨细胞（ OC）活性及OB雌激素受体（ ER）α及βmRNA表达的影响，从细胞水平阐释其防治骨质疏松症的作用机制。方法：分别选取1 d和5 d SD大鼠分离培养OB和OC，并建立OB-OC共育体系；在共育体系中，用MTT法检测低浓度（0.05、0.1、0.5、1 mg／L）、中浓度（2、5、10 mg／L）和高浓度（20、50和100 mg／L）梓醇干预下的OB增殖率，并以梓醇最佳促OB增殖浓度进行后续实验，实验分为对照组和梓醇组。 pNPP法检测各组OB的ALP活性；光镜观察OC骨吸收陷窝数目；重氮盐法检测OC抗酒石酸酸性磷酸酶（ TRAP）的活性；RT-PCR方法检测OB ERα及ERβmRNA的表达。结果：在OB-OC共育体系中，0.05～2 mg／L梓醇各组中OB的增殖率显著高于对照组（P＜0.01），且0.05 mg／L梓醇组促进OB增殖的能力明显高于其它浓度组（P＜0.01），5～100 mg／L梓醇各组OB的增殖率与对照组比较无显著差异（ P＞0.05）。0.05 mg／L梓醇组的ALP水平高于对照组（ P＜0.05），并在作用48、72和96 h后均对OC骨吸收陷窝数及TRAP有明显抑制作用（ P＜0.01）；0.05 mg／L梓醇组OB中ERαmRNA的表达与对照组相比差异无统计学意义（ P＞0.05），而ERβmRNA的表达显著高于对照组（ P＜0.05）。结论：梓醇在共育体系中可提高OB增殖和成骨活性，抑制OC活性并上调OB中ERβmRNA的表达。%[ ABSTRACT ] AIM: To investigate the effect of catalpol on the activity of osteoblasts ( OB ) and osteoclasts ( OC) , and OB estrogen receptor ( ER) α/βmRNA expression in the OB-OC co-culture system.METHODS: OB and OC were isolated from the SD rats of 1 and 5 days old.In the OB-OC co-culture system, different concentrations of catalpol including low dosage (0.05 , 0.1, 0.5 and 1 mg/L), middle dosage (2, 5 and 10 mg/L), and high dosage (20, 50 and 100 mg/L) were added into the culture medium to detect the changes of OB proliferation by MTT assay.The catalpol at maximal dosage was added to OB section to detect the alkaline phosphatase ( ALP) activity of OB by pNPP method.The mRNA expression of ERα/βin the OB treated with catalpol in the co-culture system was detected by RT-PCR.The catalpol at maximal dosage was added to OC group to detect the activity of OC by microscopy and tartrate-resistantacid phosphatase ( TRAP) activity detection.RESULTS:In 0.05~2 mg/L catalpol groups, the proliferation of OB was significantly in-creased as compared with control group in the co-culture system, and it reached the maximum value when catalpol was at 0.05 mg/L, while in 5~100 mg/L catalpol groups, the proliferation of OB was not increased.The ALP activity of OB in 0.05 mg/L catalpol group was higher than that in control group.The catalpal at 0.05 mg/L promoted the mRNA expression of ERβin OB in the co-culture system, but did not increase the mRNA expression of ERαas compared with control group. Catalpol at 0.05 mg/L obviously inhibited the bone resorption and the TRAP activity in OC.CONCLUSION: Catalpol stimulates the proliferation and activity of OB, inhibits the bone resorption and activity of OC, and increases the mRNA ex-pression of ERβin OB in the OB-OC co-culture system, suggesting that high mRNA expression of ERβmay be the regula-tory pathway of catalpol in response to bone metabolism.