特立帕肽通过Wnt3a/β-catenin通路对高糖环境下成骨细胞分化的影响

Effect of teriparatide on osteoblast differentiation in high glucose environment through Wnt3a/β-catenin pathway

目的研究特立帕肽(TPTD)通过Wnt3a/β-catenin通路对高糖环境下MC3T3-E1细胞向成骨分化的影响。方法实验分为5组:低糖组、低糖+TPTD组、高糖组、高糖+TPTD组、高糖+TPTD+G244-LM组。Calcein-AM染色及CCK-8法检测细胞增殖活性,碱性磷酸酶(ALP)及茜素红s染色观察细胞矿化结节形成情况,免疫荧光检测分析肌动蛋白形成情况,Real-time PCR检测Wnt3a、β-catenin、Tcf1、OPG、COLⅠmRNA表达情况。结果TPTD对高糖条件下MC3T3-E1细胞的增殖活性无显著影响。低糖+TPTD组细胞ALP染色区域、蛋白活性及茜素红s染色面积均高于其他四组(P<0.05),且高糖组低于低糖组(P<0.05),高糖+TPTD组高于高糖组和高糖+TPTD+G244-LM组(P<0.05)。低糖+TPTD组细胞骨架最清晰;高糖组和高糖+TPTD+G244-LM组细胞骨架清晰程度均低于高糖+TPTD组。低糖+TPTD组细胞Wnt3a、β-catenin、Tcf1、OPG、COLⅠmRNA水平最高(P<0.05);低糖组各基因mRNA水平均高于高糖组(P<0.05);高糖+TPTD组细胞各基因mRNA水平均高于高糖组和高糖+TPTD+G244-LM组(P<0.05)。结论高糖抑制成骨细胞分化,TPTD通过调控Wnt3a/β-catenin通路促进高糖环境下成骨细胞分化。

Objective To investigate the effect of teriparatide(TPTD)on the generation of MC3T3-E1 cells towards osteogenic differentiation via the Wnt3a/β-catenin pathway in a high-glucose environment.Methods The experiment was divided into five groups:low glucose group,low glucose+TPTD group,high glucose group,high glucose+TPTD group,high glucose+TPTD+Wnt3a inhibitor G244-LM group.Cell proliferation activity was detected by Calcein-AM and CCK-8 assay,cell mineralized nodule formation was observed by ALP and alizarin red staining,and actin formation was analyzed by immunofluorescence assay.Real-time PCR was performed to detect Wnt3a,β-catenin,Tcf1,OPG and COLⅠmRNA expression.Results TPTD had no significant effect on the proliferative activity of MC3T3-E1 cells under high glucose condition.The ALP staining area,protein activity and alizarin red staining area of the cells in the low glucose+TPTD group were higher than those in the other four groups(P<0.05);the high glucose group was lower than the low glucose group(P<0.05);the high glucose+TPTD group was higher than the high glucose group and the high glucose+TPTD+G244-LM group(P<0.05).The cytoskeleton in the low glucose+TPTD group was the clearest;the cytoskeleton was less clear in both the high glucose and high glucose+TPTD+G244-LM groups than in the high glucose+TPTD group.Genes such as Wnt3a,β-catenin,Tcf1,OPG and COLⅠhad the highest mRNA levels in the cells of the low glucose+TPTD group(P<0.05);the mRNA levels of all genes were higher in the low glucose group than thosein the high glucose group(P<0.05);the mRNA levels of all genes in the cells of the high glucose+TPTD group were higher than those in the high glucose group and the high glucose+TPTD+G244-LM group(P<0.05).Conclusion High glucose inhibited osteoblast differentiation,and TPTD promoted osteoblast differentiation in high glucose environment by regulating Wnt3a/β-catenin pathway.