IL-33稳定沉默后通过削弱AKT磷酸化抑制间充质干细胞增殖

Stable silencing of IL-33 inhibits mesenchymal stem cell proliferation by attenuating of AKT phosphorylation

目的:通过慢病毒载体沉默IL-33在间充质干细胞(MSCs)中的表达,探讨IL-33基因沉默后对MSCs增殖、凋亡的影响及其作用机制。方法:通过构建IL-33基因的shRNA慢病毒载体转染MSCs;应用RT-PCR及免疫印迹方法检测IL-33在MSCs中的表达情况及IL-33基因的沉默效果;通过Annexin V/PI流式细胞术检测细胞凋亡情况,进而检测IL-33基因沉默后对MSCs增殖、凋亡的影响,并通过免疫印迹检测AKT磷酸化水平。结果:细胞转染及免疫印迹实验表明IL-33 shRNA慢病毒载体构建成功,并干扰了IL-33在MSCs中的表达;稳定沉默IL-33基因后,MTT细胞生长实验表明MSCs增殖能力减弱(P<0.05);流式细胞技术和免疫印迹技术检测显示IL-33基因沉默后可提高MSCs对外界凋亡诱导物的敏感性,且引起细胞survivin基因表达的下调。结论:IL-33基因可能与MSCs增殖和抗凋亡有关,进而参与MSCs的发育和存活,IL-33稳定沉默后通过削弱AKT磷酸化可抑制MSCs增殖。

Objective:To research the effect and mechanism of IL-33 gene silencing on the proliferation and apoptosis of mesenchymal stem cells(MSCs)using lentiviral vectors.Methods:MSCs were transfected by constructing shRNA lentiviral vector of IL-33 gene,RT-PCR and Western blot were used to detect the expression of IL-33 in MSCs and the silencing effect of IL-33 gene.Apoptosis was detected by Annexin V/PI flow cytometry,and then the effect of IL-33 gene silencing on the proliferation and apoptosis of MSCs was detected,and the level of AKT phosphorylated was detected by immunoblotting.Results:Cell transfection and immunoblot experiments showed that the construction of the IL-33 shRNA lentiviral vector was successful and interfered with the expression of IL-33 in MSCs;after stable silencing of the IL-33 gene,MTT cell growth experiments showed that MSCs had reduced proliferation ability(P<0.05).Flow cytometry and immunoblot detection showed that the silencing of IL-33 gene could increase the sensitivity of MSCs to external apoptosis inducers,and also caused the down-regulation of cell survivin gene expression.Conclusion:IL-33 gene may be related to the proliferation and anti-apoptosis of MSCs,and then participate in the development and survival of MSCs,IL-33 can inhibit MSCs proliferation by attenuating AKT phosphorylation after stable silencing.