中文摘要：

目的探究R脊椎蛋白1（RSPO1）在成骨分化中的作用及机制。方法利用xCELLigence系统研究RSPO1对C2C12细胞增殖及细胞活力的影响。通过磷酸酶检测试剂盒检测ALP活性，ELISA法检测护骨因子的表达，通过双荧光素酶报告基因检测系统，利用TOPflash T细胞因子（TCF）报告质粒检测Wnt/β-联蛋白（β-catenin）信号通路活性。利用蛋白印迹法检测β-catenin蛋白累积情况。采用t检验进行统计学分析。结果RSPO1对C2C12细胞增殖及细胞活力无影响。RSPO1单独处理上调ALP活力（2.85±0.08和1.74±0.21，t=3.014，P〈0.05）及护骨因子表达能力（1.29±0.13和1.0±0.21，t=3.348，P〈0.05）较弱，而RSPO1及Wnt3A协同处理可显著增强ALP活力（81.37±5.08和1.74±0.21，t=31.31，P〈0.01）并上调护骨因子表达（5.26±0.60和1.00±0.21，t=6.99，P〈0.01）。RSPO1协同Wnt3A激活TCF活性并诱导β-catenin蛋白积累（3.28±0.18和1.00±0.21，t=10.94，P〈0.05）。但是，单独RSPO1对TCF的活性及β-catenin积累无影响（1.25±0.08和1.0±0.21，t=2.225，P〉0.05）。结论RSPO1可协同Wnt3A激活Wnt/β-catenin信号通路，诱导细胞成骨分化，提示RSPO1有望应用于RA的治疗。

英文摘要：

Objective To investigate the role and mechanism of RSPO1 in osteoblasts differentiation.Methods The xCELLigence system was used to study the toxicity and role of RSPO1 on the C2C12 cells proliferation. Alkaline phosphatase （ALP） activity was measured by using a phosphatase detection kit. The expression of osteoprotegerin （OPG） was determined using enzyme-linked immunosorbent assay （ELISA）. Wnt/β-catenin signaling was evaluated using the TOPflash T cell factor （TCF） luciferase reporter assay. Western blotting assay was performed to confirm the accumulation of β-catenin protein. T test was used for statistical analysis.Results RSPO1 had no effect on the C2C12 cells proliferation, and it produced no toxicity to C2C12 cells. RSPO1 alone resulted in a slight increase in the activity of ALP （2.85±0.08 vs 1.74±0.21, t=3.014, P〈0.05） and the expression of OPG （1.29±0.13 vs 1.00±0.21, t=3.348, P〈0.05） , whereas the combination of RSPO1 and Wnt3A significantly amplified ALP activity （81.37±5.08 vs 1.74±0.21, t=31.31, P〈0.01） and OPG protein expression （5.26±0.60 vs 1.00±0.21, t=6.99, P〈0.01）. RSPO1 synergized with Wnt3A to activate TCF activity and induce accumulation of β-catenin （3.28±0.18 vs 1.00±0.21, t=10.94, P〈0.05）. However, RSPO1 alone had no effect on the TCF activity and β-catenin accumulation （1.25±0.08 vs 1.00±0.21, t=2.225, P〉0.05） .Conclusion Our study has revealed that RSPO1 synergized with Wnt3A in activating Wnt/β-catenin signaling pathway to induce osteoblasts differentiation, which demonstrate the therapeutic potential of RSPO1 for RA.