左归丸、右归丸含药血清对大鼠脂肪干细胞成骨分化的影响

Effects of Zuogui Pill and Yougui Pill Containing Serum on Osteogenic Differentiation of Rat Adipose Stem Cells

目的比较左归丸、右归丸对大鼠脂肪干细胞成骨性分化的影响并探讨相关机制。方法将30只SD大鼠随机分为左归丸组、右归丸组和对照组各10只,左归丸组、右归丸组灌胃相应药物的浓度均为7.55g/(kg·d),对照组灌胃等量生理盐水,连续灌胃3天后制备左归丸、右归丸和对照含药血清。分离大鼠脂肪间充质干细胞,分为左归丸组、右归丸组和对照组,加入相应的体积分数10%的含药血清诱导培养7天后进行碱性磷酸酶(ALP)染色,诱导28天后进行茜素红染色,诱导培养10天检测各组细胞Wnt3a、Lef-1、β-catenin蛋白表达。结果各组细胞ALP染色均为阳性,右归丸组细胞大面积深染,明显强于左归丸组及对照组。各组细胞茜素红染色均为阳性,右归丸组的矿化结节在数量和大小上优于左归丸组及对照组。左归丸组及右归丸组β-catenin、Lef-1蛋白表达均较对照组升高(P<0.05),右归丸组Wnt3a蛋白表达亦较对照组升高(P<0.05);并且右归丸组β-catenin、Lef-1、Wnt3a蛋白表达均高于左归丸组(P<0.05)。结论右归丸及左归丸均促进大鼠脂肪干细胞成骨性分化,且右归丸作用更为显著,其机制可能与上调β-catenin、Lef-1及Wnt3a蛋白,激活经典Wnt通路有关。

Objective To compare the effects of Zuogui Pill[Left-Restoring（Kidney Yin）Pill]and Yougui Pill[RightRestoring（Life Gate）Pill]on osteogenic differentiation of rat adipose stem cells and study the related mechanisms.Methods Thirty SD rats were randomized into the Zuogui Pill group,Yougui Pill group and control group,with 10in each.The Zuogui Pill group and Yougui Pill group were gavaged with 7.55g/（kg·d）corresponding drugs.The control group was gavaged with same amount of normal saline.The containing serum was prepared after 3-day intragastric administration.The adipose mesenchymal stem cells were separated from the rats in the control group and divided into the Zuogui Pill group,Yougui Pill group and control group,with corresponding Zuogui Pill serum,Yougui Pill serum,or normal saline serum added in respectively.The alkaline phosphatase（ALP）staining was conducted after 7days of containing serum induced culture.The alizarin red staining was conducted after 28days of containing serum induced culture.The protein expression of Wnt3a,Lef-1andβ-catenin was detected after 10days of containing serum induced culture.Results The ALP staining of cells in each group were positive.A large area of deep-dyed cells could be seen in the Yougui Pill group as compared with the Zuogui Pill group and control group.The alizarin red staining of cells in each group were positive.The number and size of mineralized nodules in the Yougui Pill group was superior to that in the Zuogui Pill group and control group.Comparing with the control group,the protein expressions of Lef-1andβ-catenin were significantly increased in the Zuogui Pill group and Yougui Pill group（P〈0.05）.Comparing with the control group,the protein expression of Wnt3awas significantly increased in the Yougui Pill group（P〈0.05）.The protein expressions of Lef-1,β-catenin and Wnt3ain the Yougui Pill group were significantly higher than those in the Zuogui Pill group（P〈0.05）.Conclusion Both Yougui Pill and Zuogui Pill can promote the osteogenic differentiation of rat adipose stem cells,but Yougui Pill shows a greater effect.Its mechanisms may be relevant to upregulating proteins of Lef-1,β-catenin and Wnt3aand activating Wnt signaling pathway.