复方补肾活血颗粒含药血清经β-catenin/TRIB3调控人骨髓间充质干细胞成骨成脂分化  

Serum Containing Compound Kidney-invigorating Granule Regulates the Osteogenic and Adipogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells Through theβ-Catenin/Tribbles Pseudokinase 3 Pathway

作　　者：胡芷苜 王久香 江渟 HU Zhimu;WANG Jiuxiang;JIANG Ting(Graduate School of Anhui University of Chinese Medicine,Anhui Hefei 230012,China;The First Affiliated Hospital of Anhui University of Chinese Medicine,Anhui Hefei230031,China)

机构地区：[1]安徽中医药大学研究生院,安徽合肥230012 [2]安徽中医药大学第一附属医院,安徽合肥230031

出　　处：《安徽中医药大学学报》2024年第5期78-85,共8页Journal of Anhui University of Chinese Medicine

基　　金：安徽省重点研究与开发计划项目(202104j07020010);安徽省高等学校自然科学研究重点项目(2022AH050510)。

摘　　要：目的探讨复方补肾活血颗粒(Compound Kidney-invigorating Granule,CKG)含药血清基于β-catenin/tribbles假激酶3(tribbles pseudokinas 3,TRIB3)调控人骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨成脂分化的作用机制。方法纯化原代BMSCs;BMSCs转染β-catenin-siRNA,成骨诱导后,碱性磷酸酶(alkaline phosphatase,ALP)染色、茜素红染色观察BMSCs成骨分化情况,qPCR法、Western blot法分别检测β-catenin、TRIB3、骨桥蛋白(osteopontin,OPN)、Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)、ALP mRNA及蛋白表达水平;Western blot法检测CKG对BMSCs中β-catenin及TRIB3蛋白表达水平的影响;BMSCs转染β-catenin-siRNA,CKG含药血清培养后,ALP染色、茜素红染色及油红O染色观察BMSCs成骨成脂分化情况,qPCR法、Western blot法分别检测β-catenin、TRIB3、OPN、RUNX2、过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptorγ,PPARγ)、adiponectin mRNA及蛋白表达水平。结果BMSCs转染β-catenin-siRNA后,ALP活力金氏单位值显著降低(P<0.05),矿化结节生成减少,TRIB3、OPN、RUNX2、ALP mRNA及蛋白表达水平降低(P<0.05);CKG能促进BMSCs中β-catenin、TRIB3蛋白的表达(P<0.05)。BMSCs转染β-catenin-siRNA并用CKG含药血清干预后,5%含药血清+siRNA组细胞中钙钴染色结块与红色钙化结节数量多于0%含药血清组,低于5%含药血清组;5%含药血清+siRNA组细胞中脂肪粒数量多于5%含药血清组,低于0%含药血清组;5%含药血清+siRNA组TRIB3、OPN、RUNX2 mRNA和蛋白表达水平,ALP活力金氏单位值显著高于0%含药血清组(P<0.05),低于5%含药血清组和5%含药血清+阴性对照组(P<0.05);5%含药血清+siRNA组中PPARγ、adiponectin mRNA和蛋白表达水平低于0%含药血清组(P<0.05),高于5%含药血清组和5%含药血清+阴性对照组(P<0.05)。结论内源性敲低β-catenin抑制BMSCs的成骨分化能力,降低TRIB3的表达;CKG含药血清促进BMSCs中β-catenin�Objective To investigate the mechanism of action of serum containing Compound Kidney-invigorating Granule(CKG)in regulating the osteogenic and adipogenic differentiation of human bone marrow mesenchymal stem cells(BMSCs)through theβ-catenin/tribbles pseudokinase 3(TRIB3)pathway.Methods Primary BMSCs were purified.BMSCs were transfected withβ-catenin-siRNA,and after osteogenic induction,alkaline phosphatase(ALP)staining and alizarin red staining were used to observe the osteogenic differentiation of BMSCs;qPCR and Western blot were used to measure the mRNA and protein expression levels ofβ-catenin,TRIB3,osteopontin(OPN),Runt-related transcription factor 2(RUNX2),and ALP;Western blot was used to observe the effect of CKG on the protein expression levels ofβ-catenin and TRIB3 in BMSCs.After BMSCs were transfected withβ-catenin-siRNA and cultured with CKG-containing serum,ALP staining,alizarin red staining,and oil red O staining were used to observe the osteogenic and adipogenic differentiation of BMSCs,and qPCR and Western blot were used to measure the mRNA and protein expression levels ofβ-catenin,TRIB3,OPN,RUNX2,peroxisome proliferator-activated receptor gamma(PPARγ),and adiponectin.Results After BMSCs were transfected withβ-catenin-siRNA,there were significant reductions in ALP activity(P<0.05),mineralized nodules,and the mRNA and protein expression levels of TRIB3,OPN,RUNX2,and ALP(P<0.05).CKG promoted the expression ofβ-catenin and TRIB3 in BMSCs(P<0.05).After BMSCs were transfected withβ-catenin-siRNA and cultured with CKG-containing serum,the 5%CKG-containing serum+siRNA group had significantly higher numbers of calcium-cobalt staining nodes and red calcified nodules than the 0%CKG-containing serum group,as well as significantly lower numbers than the 5%CKG-containing serum group;the 5%CKG-containing serum+siRNA group had a significantly higher number of lipid droplets in cells than the 5%CKG-containing serum group and a significantly lower number than the 0%CKG-containing serum group;the 5%CKG-cont

关 键 词：骨质疏松症 复方补肾活血颗粒 骨髓间充质干细胞 Β-CATENIN TRIB3 成骨成脂分化 

分 类 号：R285.5[医药卫生—中药学] R681[医药卫生—中医学]