蛋白激酶CβⅡ负调控小鼠骨髓间充质干细胞的成骨分化

Protein kinase CβⅡ negatively regulates the osteoblast differentiation of mouse bone marrow-derived mesenchymal stem cells

目的观察蛋白激酶C(PKC)各亚型在小鼠骨髓间充质干细胞(BMSCs)成骨分化中的作用。方法Western blot检测小鼠BMSCs成骨分化中PKC各亚型的活化情况;利用PKC阻断剂GFX分析PKC在BMSCs体外成骨分化中的作用;碱性磷酸酶(ALP)试剂盒检测成骨早期分化指标ALP的活性;茜素红染色检测BMSCs体外矿化能力;实时荧光定量聚合酶链反应(PCR)检测成骨分化相关基因Runx2、Ⅰ型骨胶原a1(ColⅠa1)和ALP的表达情况。结果小鼠BMSCs成骨分化过程中,仅检测到PKCβⅡ亚型活化,其他亚型均无明显活化。利用PKC阻断剂GFX抑制PKCβⅡ活化后,茜素红染色结果显示BMSCs的成骨分化过程受到促进;PCR结果显示成骨分化相关基因Runx2、ColⅠa1和ALP的mRNA水平均有不同程度增加;ALP活性检测结果也显示BMSCs的成骨分化过程受到促进。结论PKCβⅡ可负调控小鼠BMSCs的成骨分化。

Objective To observe the effect of different subtype of protein kinase C（ PKC） on the osteoblast differentiation of mouse bone marrow-derived mesenchymal stem cells（ BMSCs）. Methods Western blot was used to detect the activity of different PKC subtype in the osteoblast differentiation of BMSCs. The PKC-specific inhibitor GFX was used to analyze the effect of PKC activation on the osteoblast differentiation of BMSCs in vitro. The alkaline phosphatase（ ALP） activity which was early marker of osteoblast differentiation was detected by ALP activity kit. Alizarin-red（ ARS） staining was used to investigate the mineralization potentials of BMSCs in vitro. The expression of osteoblast differentiation-related genes Runx2,Collagen typeⅠ a1（ ColⅠa1） and ALP was examined by real time fluorescent quantitation polymerase chain reaction（ PCR）. Results Only the activation of PKCβⅡ was found in the osteoblast differentiation of BMSCs. When PKCβⅡ activity was inhibited,ARS staning results showed that the osteoblast differentiation of BMSCs was promoted. PCR results showed that mRNA levels of various osteoblast differentiation markers increased to different extent. ALP activity assay results also showed that the osteoblast differentiation of BMSCs was promoted. Conclusion PKCβⅡ negatively regulates the osteoblast differentiation of BMSCs.