糖化低密度脂蛋白对小鼠成骨细胞增殖分化和低密度脂蛋白相关蛋白5、DKKl基因表达的影响

Effects of glycated low density lipoprotein on the proliferation and differentiation and expression of low- density lipoprotein receptor-related protein-5 mRNA, dickkopf-1 mRNA in mouse osteoblasts

目的研究不同浓度糖化低密度脂蛋白（糖化LDL）对小鼠成骨细胞MC3T3-E1增殖分化及低密度脂蛋白相关蛋白5（LRP5）、DKKl基因表达的可能影响，探讨Wnt信号通路是否参与了糖化LDL对成骨细胞增殖分化的调节。方法在小鼠成骨细胞株MC3T3-E1培养液中加入0．1mg／ml的不同浓度的糖化LDL，分别培养24、48、72h后应用CCK-8检测其对成骨细胞增殖的影响，采用ELISA法检测成骨细胞骨钙素水平：采用reahimePCR方法检测糖化LDL对成骨细胞LRP5、DKKl基因表达的影响。结果在LDL糖化浓度为5．3％24h时，明显抑制成骨细胞的增殖，与对照组比较，差异具有统计学意义（P〈0．01），LDL糖化越高．对成骨细胞增殖的抑制越明显．在一定的时间和浓度范围内糖化LDL对成骨细胞增殖的作用呈剂量及时间依赖性：与对照组比较，糖化LDL明显抑制成骨细胞中的骨钙素表达，差异具有统计学意义（P〈0．01）．糖化LDL对成骨细胞骨钙素表达的作用在一定程度上具有剂量及作用时间依赖性；5．3％糖化LDL在48h时明显下调成骨细胞中的LRP5mRNA表达水平，显著上调DKKlmRNA表达水平，与对照组比较．差异具有统计学意义（P〈0．01）。结论糖化LDL显著抑制成骨细胞的增殖和分化，这可能与Wnt信号通路中LRP5及DKKl基因表达有关。

Objective The aim of this study was to investigate the possible effect of glycated low density lipoprotein （LDL） on the proliferation, differentiation, and expression of low-density lipoprotein receptor-related protein 5 （LRPS） mRNA, dickkopf-1 （ DKK1 ） mRNA in mouse osteoblasts （ MC3T3-E1 cells ）. Whether the Wnt signaling pathway was involved in the above process was explored else. Methods Mouse MC3T3-E1 cells were cultured with various concentrations of glycated LDL （ glycated LDL level was 2.4% , 5.3 % , 8.7 % , 13.9 % ） for 24, 48, 72 h. Proliferation of MC3T3-E1 cell was measured by CCK-8, the osteocalcin level in the medium was determined by ELISA and the expression of LRP5 mRNA, DKK1 mRNA was measured by realtime PCR. Results After cells being incubated with 5.3% of glycated LDL for 24 h, the inhibition of MC3T3-E1 cells was more marked than that in control group （ P〈0.01 ）. The higher glycated LDL level, the severer the inhibition. The effect of LDL on the MC3T3-E1 cell proliferation was time- and dose-dependent under certain conditions. Osteocalcin level in cell culture fluid with glycated LDL was lowered significantly compared with control group. Meanwhile, the expression of DKK1 mRNA was increased significantly but expression of LRP5 mRNA decreased （P〈0.01） in MC3T3-E1 cells cultured with 5.3 % of glycated LDL for 24 h. Conclusions Proliferation and differentiation of the osteoblasts in mice can be inhibited significantly by glycated LDL. The possible mechanism in the role played by glycated LDL on the proliferation and differentiation in MC3T3-E1 cells seems to be related to expression of LRP5 mRNA, DKK1 mRNA in the Wnt signaling pathway.