摘要
目的研究apelin对人成骨细胞凋亡的作用。方法用茜素红染色观察原代培养的人成骨细胞矿化结节的形成。碱性磷酸酶（ALP）用ELISA检测；骨钙素（OC）用放射免疫测定法测定；I型胶原用ELISA法检测。用ELISA法检测细胞凋亡。Bcl-2，Bax，caspase-3，cytochromeC，p-Akt，Akt用Westernblot检测。P13．Kp850r用免疫沉淀法检测。用小分子RNA干扰技术（siRNAs）抑制APJ表达，联合P13-K信号转导阻断剂LY294002及Akt信号转导阻断剂HIMO干预，以观察Apelin对成骨细胞凋亡的作用及信号转导。结果Apelin能抑制无血清诱导的人成骨细胞凋亡，siRNAs转染阻断APJ表达可消除此效应。Apelin干预可诱导人成骨细胞Bcl-2蛋白质表达，抑制Bax蛋白质表达和CytochromeC的分泌及caspase一3的裂解活化。Apelin可诱导人成骨细胞P13．K及Akt磷酸化；siRNAs转染沉默APJ可消除此效应；P13-K信号转导阻断剂LY294002或Akt阻断剂HIMO能消除Apelin对人成骨细胞Akt的活化作用；LY294002或HIMO也能阻断Apelin对人成骨细胞抑制凋亡作用。结论Apelin可抑制人成骨细胞的凋亡；Apelin通过APJ／P13-K／Akt信号通路介导参与对成骨细胞凋亡的调节。
Objective To investigate the action of apelin on apoptosis of human osteoblasts. Methods Alizarin red staining was used for assessment of human osteoblasts matrix mineralization. Alkaline phosphatase （ALP） activity was measured using ALP ELISA kit, osteocalcin （OC） was measured by RIA, Procollagen I C-terminal propeptide （PICP） in the cell-conditioned medium was measured using an ELISA Kit. Cell apoptosis was analyzed by ELISA. The expression of Bcl-2, Bax, caspase-3, cytochrome C, p-Akt, Akt were measured using Western blot, and the expression of PI3-Ki- nase p85α was detected by immuno-precipitation. RNA interference was used to down-regulate the expression of APJ in human osteoblasts. PI3-K inhibitor LY294002 and Akt inhibitor HIMO were also used for the signaling pathway study. Results Apelin inhibited human osteoblasts apoptosis, suppression of APJ with siRNA abolished apelin＇ effects on human osteoblasts apoptosis. Western blot analysis showed that apelin increased Bcl-2 protein and decreased Bax protein expres- sion, and also blocked the release of cytochrome c and activation of caspase-3. Apelin activated the phosphorylation of phosphatidylinositol 3-kinase （PI3-K） and Akt; these effects were blocked by suppression of APJ with siRNA; pretreat- ment of osteoblasts with PI3-kinase inhibitor LY294002 or Akt; inhibitor HIMO abolished apelin-induced activation of Akt. Furthermore, LY294002 or HIMO abolished apelin＇s anti-apoptotic activity in human osteoblasts. Conclusion Apelin could suppress serum deprivation-induced apoptosis of human osteoblasts and the anti-apoptotic action is mediated via the APJ/PI-3 kinase/Akt signaling pathway.
出处
《中华骨质疏松和骨矿盐疾病杂志》
2008年第1期39-45,共7页
Chinese Journal Of Osteoporosis And Bone Mineral Research
基金
国家自然科学基金资助项目（30200322,30400218）及湖南省杰出青年基金（03JJY1005）
