诱导成骨分化过程中BMP与WNT信号通路串话机制

Crosstalk mechanism of BMP signaling pathway and Wnt signaling pathway in osteogenic differentiation

目的探究小鼠骨髓间充质干细胞成骨分化过程中,BMP-2/Smad信号通路与Wnt/β-catenin通路的串话机制。方法将预先准备好的重组人骨形态发生蛋白-2(rh BMP-2)质粒转染至C3H10T1/2细胞,利用G418抗药性筛查转染成功的细胞。将实验细胞分为转染组、空白组。通过ELISA检测各组细胞上清液中的BMP-2含量,采用Western blot和RT-PCR方法检测两组中Smad泛素化调节因子2(Smurf2)、β-链蛋白(β-catenin)、磷酸化β-链蛋白(pβ-catenin)、转录因子RUNX2、淋巴增强因子1(LEF1)、Axin2、糖原合成激酶-3β(GSK-3β)等关键细胞因子的表达水平。结果与空白组比较,转染组BMP-2浓度升高并维持在较高水平(P<0.05),β-catenin mRNA的表达上调(P<0.05)和磷酸化水平增加(P<0.01),LEF1蛋白合成上调(P<0.05),Smurf2蛋白表达减少(P<0.05),RUNX2的mRMA表达上调(P<0.01)。结论在成骨细胞分化过程中,BMP-2/Smad信号通路与Wnt/β-catenin通路存在串话机制,增加β-catenin的表达水平,促进β-catenin的磷酸化。

Objective To investigate the crosstalk mechanism between BMP- 2/Smad signaling pathway and Wnt/β -catenin pathway in osteogenic differentiation of mouse bone marrow mesenchymal stem cells （MSCs）. Methods The recombinant human BMP-2 （rhBMP- 2） plasmid was transfected into C3H10T1/2 cells. G418 resistance was applied for transfection screening. The experimental cells were divided into transfeeted group and blank group （ no plas- mid）. The concentration of BMP -2 in medium supernatant was detected by BMP -2 ELISA kit. Western blot and RT - PCR were used to assessed the expression levels of key cytokines, such as Smurf2,β - catenin, phosphorylation β - cate- nin （p β - catenin）, RUNX2, LEF1, Axin and GSK3β in the both 2 groups. Results Compared with the blank group, the concentration of BMP - 2 in transfected group was significantly increased and maintained at a high level （ P 〈 0.05 ）. The mRNA expression and phosphorylation level of β - catenin were significantly increased in transfected group （ P 〈 0. 05 ）. The mRNA expression and protein synthesis of LEF1 were significantly up - regulated in transfected group （ P 〈 0. 05 ）. The mRNA expression and protein synthesis of Smurf2 were significantly reduced in transfeeted group （P 〈 0. 05 ）. The mRNA expression of GSK3β and Axin2 were down - regulated in transfected group （ P 〈 0. 05 ）. Conclusion In the osteogenic differentiation process, crosstalk mechanisms exists between BMP -2/Smad signaling pathway and Wntβ - catenin signaling pathway. It increases the expression ofβ - eatenin and promotes the phosphorylation of β - ecatenin.