奥氮平对骨髓间充质干细胞成脂分化的作用及机制

Effects and mechanism of olanzapine on the adipose differentiation of mouse bone marrow mesenchymal stem cells

目的探讨第2代抗精神病药物(SGA)奥氮平(olanzapine)对小鼠骨髓间充质干细胞(BMSCs)体外脂肪分化的影响及其作用机制。方法培养小鼠BMSCs,MTT法检测不同浓度奥氮平对BMSCs增殖的影响;通过油红O染色观察细胞形态,Western blotting检测脂肪分化标志基因蛋白αP2和C/EBPβ的表达,Real-time PCR检测成脂相关基因Leptin、C/EBPα和TNF-α的mRNA表达情况;同时Western blotting检测Akt信号通路上下游相关分子的表达水平及磷脂酰肌醇3激酶(PI3K)/Akt特异性阻断剂对小鼠BMSCs脂肪分化的影响。结果 MTT法结果显示,20μmol/L奥氮平对BMSCs的毒性最小;油红O染色可见加入奥氮平的细胞内脂肪滴明显多于对照组;Western blotting结果显示,αP2和C/EBPβ的表达水平较对照组上升了36%(P<0.01)和25%(P<0.05);Realtime PCR结果显示,Leptin、C/EBPα和TNF-α的表达水平较对照组分别上升68%(P<0.001)、79%(P<0.01)和60%(P<0.01),均具有统计学意义;Western blotting检测结果显示,p-Akt的含量明显高于对照组,磷酸化糖原合成激酶3β(p-GSK-3β)的表达随着p-Akt表达的增加逐渐减少;加入PI3K/Akt特异性阻断剂(LY294002)后αP2和C/EBPβ的表达受到抑制,细胞中的脂肪滴也明显减少。结论奥氮平可能通过促使PI3K/Akt信号通路中Akt的磷酸化水平升高来促进小鼠BMSCs的脂肪分化。

Objective To investigate the effects of olanzapine on the adipose differentiation and mechanism of mouse bone marrow mesenchymal stem cells（ BMSCs）. Methods MTT colorimetry was used for assaying different concentrations of olanzapine on the proliferation of mouse BMSCs. The morphology of cells was observed by Oil red O staining. The expressions levels of adipocyte markers αP2 and C / EBPβ were detected by Western blotting,mRNA expression levels of the related genes of Leptin,C / EBPα and TNF-α were analyzed by Real-time PCR. The protein expression of Akt signaling pathway related molecules and effects of phosphatidylinositol 3 kinase（ PI3K） / Akt inhibitor on the differentiation of BMSCs were determined by Western blotting. Results The results of MTT colorimetry showed that20μmol / L olanzapine had minimal toxicity on BMSCs. Oil red O staining showed that intracellular lipid droplets in the experimental group were significantly more than the control group. The results of Western blotting showed that the expression levels of αP2 and C / EBPβ increased by about 36%（ P〈0. 01） and 25%（ P〈0. 05） compared with the control group respectively. The results of Real-time PCR showed that the expression levels of adipogenic genes Leptin,C / EBPαand TNF-α significantly increased than the control group,increased about 68%（ P〈0. 001）,79%（ P〈0. 01） and 60%（ P〈0. 01） respectively. The expression levels of p-Akt were significantly higher than the control group,the expression of glycogen synthase kinase 3β（ GSK-3β） with the increase of p-Akt expression gradually reduced. The expression levels ofαP2 and C /EBPβ were inhibited after BMSCs were pretreated with PI3 K /Akt inhibitor（ LY294002）. Oil red O staining results showed fat droplets in cells also decreased significantly. Conclusion Olanzapine may elevate the expression level of p-Akt of PI3 K / AKT signaling pathway to promote the adipogenic differentiation of mouse BMSCs.