β-catenin对骨形态发生蛋白9诱导间充质干细胞成骨分化的影响  

Effect of β-catenin on osteogenic differentiation of mesenchymal stem cells induced by bone morphogenetic protein 9

作　　者：林良波[1] 徐娇[2] 张晓[1] 符纯锋[1] 王志强[1] 周建武[2] 毕杨[2] 黄伟[1] 

机构地区：[1]重庆医科大学附属第一医院骨科,重庆400016 [2]重庆医科大学附属儿童医院儿科研究所干细胞实验室,重庆400014

出　　处：《中国生物制品学杂志》2014年第1期52-57,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(31070875)

摘　　要：目的探讨经典Wnt信号通路关键节点β-catenin对骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)诱导间充质干细胞(mesenchymal stem cells,MSCs)成骨分化的影响。方法用重组腺病毒介导BMP9在C3H10T1/2细胞中过表达,联用β-catenin重组腺病毒上调β-catenin的表达,并通过RNA干扰抑制β-catenin的表达。分析C3H10T1/2细胞碱性磷酸酶(alkaline phosphatase,ALP)活性的变化;RT-PCR检测细胞成骨分化相关基因骨桥蛋白(osteopontin,OPN)和骨钙蛋白(osteocalcin,OC)基因mRNA的转录水平;茜素红S染色检测细胞的钙盐沉积。结果 BMP9单独作用能诱导C3H10T1/2细胞向成骨方向分化,并增强细胞ALP活性;单独的β-catenin无成骨诱导作用,但可剂量依赖性地增强BMP9诱导的C3H10T1/2细胞的ALP活性,并促进BMP9诱导的细胞OPN和OC基因mRNA的转录水平及钙盐沉积;抑制β-catenin表达可显著降低BMP9诱导的C3H1OT1/2细胞的ALP活性(P<0.05),下调OPN和OC基因mRNA的转录水平,并抑制钙盐沉积。结论经典Wnt信号通路可能通过β-catenin协同BMP9诱导C3H10T1/2细胞成骨分化,且BMP9诱导的成骨分化可能需要通过Wnt/β-catenin途径来实现。Objective To investigate the effect of β-catenin on bone morphogenetic protein （BMP） 9-induced osteogenic differentiation of mesenchymal stem cells （MSCs） via Wnt/β-catenin signaling pathway. Methods C3H10TI /2 cells were infected with recombinant adenovirus expressing BMP9, while the essential canonical Wnt signaling mediator β-catenin was over-expressed by Ad-β-catenin, and the knock-down of β-catenin expression was achieved by adenovirus- mediated siRNA specific for β-catenin. The C3HI0TI/2 cells were analyzed for activity of alkaline phosphatase（ALP）, for transcription levels of osteopontin （OPN） and osteocalcin （OC） mRNAs by RT-PCR, and for calcium deposition by alizarin red S staining. Results BMP9 alone induced the osteogenic differentiation of C3H10T1 /2 ceils and increased the ALP activity. However, β-catenin alone induced no osteogenic differentiation, while increased the BMP9-induced ALP activity in a dose-dependent pattern, and promoted the OPN and OC mRNA transcriptions and calcium deposition induced by BMP9. The inhibition of β-catenin expression decreased the ALP activity in C3H10T1 / 2 cells induced by BMP9 （P 〈 0. 05）, down-regulated the transcription levels of OPN and OC mRNAs and inhibited the calcium deposition. Conclusion Canonical Wnt signaling pathway might act synergistically with BMP9 in induction of osteogenic differentia- tion of C3H10T1/2 ceils, and BMP9-induced osteogenie differentiation might achieve via functional canonical Wnt/ [3-catenin signaling pathway.

关 键 词：WNT Β-CATENIN 骨形态发生蛋白9 间充质干细胞 成骨分化 

分 类 号：Q254[生物学—细胞生物学]