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目的研究apelin及其受体APJ在人成骨细胞的表达和apelin对成骨细胞的作用。方法RT-PCR和Western印迹法检测apelin和APJ在人成骨细胞的表达。用apelin处理人成骨细胞,~3H掺入法(~3H-TdR)和细胞计数法测定细胞的增殖,通过测定细胞内碱性磷酸酶(ALP)活性、骨钙素分泌水平和Ⅰ型胶原的生成来分析细胞功能。Western印迹法检测信号通路的激活。通过小分子干扰核糖核酸(siRNA)技术阻断APJ,观察其对细胞增殖和信号通路的影响。结果Apelin和APJ在人成骨细胞均有表达,Apelin能促进人成骨细胞增殖,但对其ALP活性、骨钙素的分泌和Ⅰ型胶原的生成无影响。通过siRNA技术阻断APJ能消除apelin对细胞增殖的促进作用。Apelin能诱导人成骨细胞磷脂酰肌醇3激酶(PI3K)下游底物蛋白激酶B(Akt)的活化,但不能诱导c-jun N-端激酶(JNK)、p38和细胞外信号调节激酶1/2(ERK1/2)等丝裂原活化蛋白激酶(MAPK)的活化。siRNA干扰APJ对Akt的活化起抑制作用。PI3K抑制剂(LY294002)能消除apelin对细胞增殖的促进作用和Akt的活化。结论人成骨细胞表达apelin和APJ,Apelin通过APJ/PI3K/Akt途径促进人成骨细胞增殖,但对其分化无影响。
Objective To study the expression of apelin and its receptor APJ in human osteoblasts and the effect of apelin on osteoblasts. Methods RT-PCR and Western blotting were used to detect the expression of apelin and APJ in human osteoblasts. Human osteoblasts were treated with apelin, cell proliferation was measured by ~ 3H incorporation method (~ 3H-TdR) and cell counting method. The activity of alkaline phosphatase (ALP), osteocalcin and collagen type Ⅰ Generate to analyze cell function. Western blotting to detect activation of signaling pathways. APJ was blocked by small interfering RNA (siRNA) to observe its effect on cell proliferation and signal pathway. Results Apelin and APJ were expressed in human osteoblasts. Apelin could promote the proliferation of human osteoblasts, but had no effect on ALP activity, secretion of osteocalcin and type Ⅰ collagen production. Blocking APJ by siRNA technology can eliminate the promotion effect of apelin on cell proliferation. Apelin can induce the activation of Akt, a downstream substrate of human osteoblast phosphatidylinositol 3-kinase (PI3K), but not c-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinase 1 / 2 (ERK1 / 2) and other mitogen-activated protein kinase (MAPK) activation. siRNA interferes APJ with inhibiting Akt activation. PI3K inhibitor (LY294002) can eliminate apelin on the promotion of cell proliferation and activation of Akt. Conclusion Human osteoblasts express apelin and APJ. Apelin can promote the proliferation of human osteoblasts through the APJ / PI3K / Akt pathway, but have no effect on the differentiation.