摘要
目的基于Wnt/β-连环蛋白(β-Catenin)信号通路研究吴门骨密葆方含药血清促进骨髓间充质干细胞成骨分化机制。方法将大鼠骨髓间充质干细胞为正常血清组、经典诱导剂[间充质干细胞成骨诱导分化培养基(OBM)]组、吴门骨密葆方含药血清低(10 g生药/kg)和高剂量(20 g生药/kg)组、抑制剂[分泌型蛋白Dickkopf-1(DKK-1),0.1 ng·mL-1]组以及吴门骨密葆方含药血清低、高剂量+抑制剂组,成骨诱导分化后干预7 d时采用CCK8法检测细胞活力,随后采用碱性磷酸酶(alkaline phosphatase,ALP)染色法和ALP试剂盒检测不同剂量吴门骨密葆方含药血清及加入Wnt/β-Catenin信号通路特异性阻滞剂DKK-1干预的间充质干细胞ALP染色强度和活性,Western Blot测定间充质干细胞Wnt信号蛋白家族3alpha(Wnt3a)、β-Catenin、糖原合成酶激酶3β(glycogen synthase kinase 3β,GSK-3β)、破骨细胞抑制因子(osteoprotegerin,OPG)和核因子κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)蛋白表达水平。结果与正常组比较,吴门骨密葆方含药血清干预均可有效促进间充质干细胞增殖(P均<0.05),吴门骨密葆方含药血清可有效促进间充质干细胞ALP染色的强度和活性(P均<0.05),以及调控成骨分化相关的Wnt3a、β-Catenin、GSK3β、OPG和RANKL蛋白表达(P均<0.05),但是这些调控作用均可被DKK-1在一定程度上逆转。结论吴门骨密葆方含药血清诱导骨髓间充质干细胞的成骨分化,可能通过激活Wnt/β-catenin信号通路以及影响OPG/RANKL通路而实现。
Objective To study mechanism of Wumen Gumi Bao decoction containing serum in promoting mesenchymal stem cells osteogenic differentiation based on Wnt/β-catenin signaling pathway.Methods SD rat bone marrow mesenchymal stem cells were divided into the normal serum group,the classic inducer[mesenchymal stem cell Osteoblast Medium(OBM)]group,Wumen Gu Mi Bao decoction containing serum low dose(10g crude drug/kg)and high dose(20g crude drug/kg)group,the inhibitor[secreted protein Dickkopf-1(DKK-1),0.1ng·mL-1]group,and Wumen Gu Mi Bao decoction containing serum low and high dose+the inhibitor groups.CCK8 method was used to detect the activity of mesenchymal stem cells after 7 days of intervention after osteogenic differentiation.And then the ALP staining intensity and activity of mesenchymal stem cells stained with alkaline phosphatase(ALP)staining method were statistically analyzed,and these mesenchymal stem cells were intervened by the different doses of Wumen Gu Mi Bao decoction containing serum,and Wnt/β-catenin signaling pathway specific blocker DKK-1.The levels of 3αlpha(Wnt3α),β-Catenin,glycogen synthase kinase 3β(GSK 3β),osteoclastogenesis inhibitory factor(OCIF),also known as osteoprotegerin(OPG),and receptor activator of nuclear factor-κB ligand(RANKL)in the Wnt signaling protein family of mesenchymal stem cells were determined by Western Blot.Results Compared with the normal group,intervention with the different doses of Wumen Gu Mi Bao decoction containing serum could effectively promote the proliferation of mesenchymal stem cells(P<0.05);Wumen Gu Mi Bao decoction containing serum could effectively promote the intensity and activity of ALP staining in mesenchymal stem cells(P<0.05),as well as regulate the expression levels of osteogenic differentiation⁃related Wnt3α,β-Catenin,GSK3β,OPG and RANKL proteins(all P<0.05),but these regulatory effects could be reversed by DKK-1to a certain extent.Conclusion Wumen Gu Mi Bao decoction may induce the osteogenic differentiation of bone marrow mesenchymal stem cells by activating Wnt/β-catenin signaling pathway and affecting OPG/RANKL pathway.
作者
梁国强
李宇卫
沈晓峰
尤君怡
张国栋
华永庆
LIANG Guoqiang;LI Yuwei;SHEN Xiaofeng;YOU Junyi;ZHANG Guodong;HUA Yongqing(Central Laboratory,Suzhou Traditional Chinese Medicine Hospital Affiliated to Nanjing University of Chinese Medicine,Suzhou 215009,China;Department of Orthopedics and Traumatology,Suzhou Traditional Chinese Medicine Hospital Affiliated to Nanjing University of Chinese Medicine,Suzhou 215009,China;Department of Rehabilitation,Suzhou Traditional Chinese Medicine Hospital Affiliated to Nanjing University of Chinese Medicine,Suzhou 215009,China;College of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China)
出处
《长春中医药大学学报》
2024年第3期271-275,共5页
Journal of Changchun University of Chinese Medicine
基金
江苏省卫生健康委员会老年健康研究项目(LKZ2023217)
南京中医药大学自然科学基金项目(XAR2021041)
苏州市科技发展计划项目(SKY2022202)
苏州市中医骨伤重点实验室建设项目(SZS2022019)
苏州市第九批姑苏卫生重点人才项目(GSWS2022107)
苏州市卫生青年骨干人才“全国导师制”培训项目(Qngg2022023)。
