靶向抑制PI3K对PDK-1Akt信号通路调控成骨细胞分化的影响

Study of targeted inhibition of PI3K on PDK-1/Akt signal pathway regulating osteoblast differentiation

目的通过观察磷脂酰肌醇3-激酶(PI3K)特异性抑制剂LY294002在成骨细胞分化过程中的影响,探讨PI3K通过调控PDK-1/Akt信号通路对成骨细胞分化影响的分子机制。方法采用体外培养C57BL/6小鼠骨髓间充质干细胞(BMSC)。通过CCK-8检测LY294002的最大安全浓度。对照组使用成骨细胞诱导培养基(OBM)处理BMSC,干预组在OBM中添加LY294002。检测两组细胞碱性磷酸酶(ALP)活性和细胞矿化能力,并分别采用Western-Blot和RT-PCR检测PDK-1/Akt信号通路及下游蛋白和转录因子基因的表达水平。结果CCK-8结果提示,在3.2μM及以下浓度对BMSC的增殖没有影响;LY294002对成骨细胞分化的影响呈剂量依赖性(P<0.05);干预组ALP阳性细胞和细胞外基质矿化较对照组明显减少(P<0.05);Western-Blot检测结果显示,干预组p-PDK-1和p-Akt蛋白表达水平较对照组明显降低(P<0.05);RT-PCR结果提示,干预组成骨细胞相关基因ALP和骨钙素(OCN)mRNA较对照组表达明显下降(P<0.05)。结论靶向抑制PI3K可通过PDK-1/Akt信号通路明显下调成骨细胞相关基因ALP和OCN的表达,并抑制成骨细胞的分化。

Objective To observe the effect of phosphatidylinositol 3-kinase(PI3K)specific inhibitor LY294002 on osteoblast differentiation,and explore the molecular mechanism of PI3K on osteoblast differentiation by regulating PDK-1/Akt signal pathway.Methods Bone marrow mesenchymal stem cells(BMSC)of C57BL/6 mice were cultured in vitro.The maximum safe concentration of LY294002 was detected by CCK-8.BMSC was treated with osteoblast induction medium(OBM)in the control group.BMSC was treated with OBM including LY294002 in the intervention group.The alkaline phosphatase(ALP)activity and mineralization ability of the two groups were detected,and the expression levels of PDK-1/Akt signal pathway and downstream protein and transcription factor genes were detected by Western-Blot and RT-PCR,respectively.Results The results of CCK-8 showed that the concentration of 3.2μM and below had no effect on the proliferation of BMSC,but the effect of LY294002 on osteoblast differentiation in a dose-dependent manner.The number of ALP positive cells and extracellular matrix mineralization in the intervention group were significantly lower than those in the control group(P<0.05).The results of Western-Blot detection showed that the protein expression levels of p-PDK-1 and p-Akt in the intervention group were significantly lower than those in the control group(P<0.05).The results of RT-PCR showed that the related genes of alkaline phosphatase(ALP)and osteocalcin(OCN)mRNA in the intervention group were significantly lower than those in the control group(P<0.05).Conclusion Targeted inhibition of PI3K can significantly down-regulate the expression of osteoblast-related genes ALP and OCN and inhibit osteoblast differentiation through PDK-1/Akt signal pathway.