基于Wnt/β-链蛋白信号通路观察高糖环境对大鼠下颌骨骨髓基质细胞增殖的影响

Observing the Effect of High Glucose on Proliferation of Bone Marrow Stromal Stem Cells through Wnt /β-catenin Pathway

目的观察高糖环境对大鼠下颌骨骨髓基质细胞增殖的影响,并结合Wnt/β-链蛋白信号通路初步探讨高糖对骨髓基质细胞增殖的影响。方法从Wistar大鼠下颌骨中分离培养骨髓基质细胞,分别给予不同糖浓度(5.5和16.5 mmol/L)干预,采用噻唑蓝法观察骨髓基质细胞1、3、5、7 d时的增殖情况,采用流式细胞仪对高糖干预5 d细胞进行细胞周期检测,Western免疫印迹检测5 d时β-链蛋白和细胞周期蛋白D1的表达,实时定量多聚酶链反应检测淋巴增强因子-1和细胞周期蛋白D1的mRNA表达。结果噻唑蓝法检测结果显示,1 d骨髓基质细胞在两种糖浓度中的光密度值差异无统计学意义(P=0.700),3、5、7 d时16.5 mmol/L组的细胞光密度值显著低于5.5 mmol/L组(P=0.006,P=0.002,P=0.003)。5 d的细胞周期结果显示,与5.5 mmol/L组细胞相比,16.5 mmol/L组细胞可以抑制细胞从G1期向S期转化,16.5 mmol/L组细胞的增殖指数显著低于5.5 mmol/L组(P=0.014)。与5.5 mmol/L组相比,16.5 mmol/L组细胞明显抑制β-链蛋白的表达,同时Wnt/β-链蛋白通路中淋巴增强因子-1 mRNA及下游细胞周期蛋白D1 mRNA和蛋白的表达也受到明显抑制。结论高糖环境可以通过抑制Wnt/β-链蛋白信号通路中关键因子β-链蛋白、淋巴增强因子-1及下游与细胞增殖相关靶基因细胞周期蛋白D1的表达,进而抑制骨髓基质细胞的增殖。

Objective To explore the effect of high glucose on proliferation of bone marrow stromal stem cells through Wnt/β-catenin pathway.Methods Bone marrow stormal cells were obtained from the mandible of Wistar rats and stimulated with different concentrations of glucose （5.5 and 16.5 mmol/L）.Cell proliferation was evaluated with methyl thiazolyl tetrazolium assay （1,3,5,and 7 d） and cell cycle analysis by flow cytometry （5 d）.β-catenin and cyclin D1 protein levels were determined by Western blot.The mRNA expression of lymphoid enhancer binding factor-1 （LEF-1） and cyclin D1 were tested by real-time polymerase chain reaction.Results The results of methyl thiazolyl tetrazolium assay indicated that the optical density values of two different concentrations of the glucose had no statistical difference on day 1 （P =0.700）.On days 3,5,and 7,the optical density values of the 16.5 mmol/L group were significantly lower than those in the 5.5 mmol/L group （P =0.006,P =0.002,and P =0.003）.Cell cycle analysis indicated that high glucose concentration could reduced the progression from phase G1 to S,and the proliferation index values of the 16.5 mmol/L group were significantly lower than those of the 5.5 mmol/L group （P =0.014）.The β-catenin and cyclin D1 levels were lower in the 16.5 mmol/L group when compared with the 5.5 mmol/L group.High glucose condition also reduced the mRNA expressions of LEF-1 and cyclin D1.Conclusion High glucose can inhibit the proliferation of bone marrow stormal cells by suppressing the expressions of β-catenin,LEF-1,and cyclin D1 in the Wnt/β-catenin pathway.