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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 effect of olanzapine, a second generation antipsychotic drug (SGA) on the adipogenic differentiation of mouse bone marrow mesenchymal stem cells (BMSCs) in vitro and its possible mechanism. Methods BMSCs were cultured and the effects of different concentrations of olanzapine on the proliferation of BMSCs were detected by MTT assay. The morphology of BMSCs was observed by oil red O staining. The expressions of αP2 and C / EBPβ were detected by Western blotting. Real-time PCR The expressions of adiponectin, leptin, C / EBPα and TNF-α were detected by Western blotting. The expressions of upstream and downstream Akt signaling molecules and PI3K / Akt specific antagonist Effect of mouse BMSCs on adipogenic differentiation. Results The results of MTT assay showed that omeprazole at 20μmol / L had the lowest toxicity to BMSCs. Oil red O staining showed that intracellular fat droplets were more than olanzapine group. Western blotting showed that the expression of αP2 and C / EBPβ The levels of Leptin, C / EBPα and TNF-α were increased by 68% (P <0.001) compared with the control group (Realtime PCR) ), 79% (P <0.01) and 60% (P <0.01), respectively. The results of Western blotting showed that the content of p-Akt was significantly higher than that of the control group. The phosphorylated glycogen synthesis kinase 3β -GSK-3β) gradually decreased with the increase of p-Akt expression; the expression of αP2 and C / EBPβ was inhibited after the addition of PI3K / Akt specific inhibitor (LY294002), and the fat droplets in the cells were also significantly reduced. Conclusion Olanzapine may promote adipose differentiation of mouse BMSCs by increasing the phosphorylation of Akt in PI3K / Akt signaling pathway.