基于Wnt信号通路研究骨康方对过表达DKK1大鼠骨形成影响

摘要

目的 利用腺病毒过表达技术研究Wnt信号通路抑制因子Dickkopf1 (DKK1) 在大鼠骨形成中的作用并观察骨康方 (补肾健脾活血方) 对过表达DKK1转染大鼠骨形成的影响作用.方法 选取成年SD大鼠18只, 利用过表达DKK1 (Ad-DKK1) 腺病毒转染其中6只大鼠, 空载腺病毒 (Ad-EV) 转染另外6只大鼠, 用生理盐水对剩余的6只大鼠进行注射并设为对照组 (NC).转染成功后, 每组随机选3只给予骨康方干预, 另外3只给予等体积生理盐水, 观察大鼠骨密度、Wnt信号通路相关因子DKK1, LRP6, β-catenin, APC, RUNX2, OPG表达量的变化.结果 腺病毒成功转染大鼠, Ad-DKK1转染组DKK1表达量明显高于Ad-EV组及NC组, Ad-DKK1组骨密度低于Ad-EV组及NC组 (P＜0.05);②中药干预后, Ad-DKK1组、Ad-EV组、NC组大鼠骨密度升高, Wnt信号通路抑制因子DKK1、APC表达量降低, Wnt信号通路成骨相关因子LRP6, β-catenin, RUNX2, OPG表达量升高 (P＜0.05).结论 ①过表达DKK1可导致大鼠股骨骨密度降低;②骨康方干预后, 大鼠股骨骨密度升高, LRP6, β-catenin, RUNX2, OPG表达量增加, DKK1、APC表达量降低.%Objective To study the role of Wnt signaling pathway inhibitor Dickkopf1 (DKK1) in rat bone formation using adenovirus overexpression technology and to observe the effect of Gukang recipe (nourishing kidney and invigorating spleen recipe) on the bone formation of DKK1-overexpressing transfected rats. Methods Eighteen adult SD rats were selected. Among them, 6 rats were transfected with adenovirus overexpressing DKK1 (Ad-DKK1), 6 rats were transfected with empty adenovirus (Ad-EV), and 6 rats were injected with normal saline as control group (NC). After successful transfection, 3 rats in each group was randomly selected to give Gukang recipe intervention, and 3 rats were given equal volume of normal saline. The changes of bone mineral density, and Wnt signaling pathway related factors DKK1, LRP6, β-catenin, APC, RUNX2, and OPG expression were observed. Results Adenovirus was successfully transfected in rats. The expression of DKK1 in Ad-DKK1 transfection group was significantly higher than that in Ad-EV group and NC group. Bone mineral density in the Ad-DKK1 group was lower than that in the Ad-EV group and the NC group (P＜0.05). ②After Chinese herbal intervention, bone mineral density in Ad-DKK1, Ad-EV, and NC groups increased, the expression of Wnt signaling pathway inhibitors DKK1 and APC decreased, and the expression of LRP6, β-catenin, RUNX2, and OPG increased (P＜0.05). Conclusion Overexpression of DKK1 can lead to decreased femoral bone mineral density in rats. ②After intervention with Gukang recipe, femoral bone mineral density in rats increases, the expression of LRP6, β-catenin, RUNX2, and OPG increases, and the expression of DKK1 and APC declines.