论文部分内容阅读
目的探讨六溴环十二烷(HBCDs)对小鼠神经母细胞瘤细胞N2a增殖的影响以及对3个重要细胞核受体:视黄醛类X受体α(RXRα)、过氧化物酶体增殖物活化受体γ(PPARγ)、孕烷X受体(PXR)表达及其相互作用的影响。方法用不同浓度的3种非对映异构体(±)α-HBCD,(±)β-HBCD,(±)γ-HBCD处理N2a细胞,Cell counting kit-8(CCK-8)法检测HBCD对N2a的细胞毒性作用;流式细胞术检测HBCD对N2a细胞周期的影响;实时荧光定量(RT-PCR)和免疫蛋白印迹(Western blot,WB)法分别用于检测3个细胞核受体RXRα、PPARγ、PXR和下游靶基因细胞色素P450亚酶CYP3A11的mRNA及蛋白表达水平的变化;免疫共沉淀技术分析RXRα、PXR、PPARγ受体间的相互作用。结果β-HBCD对N2a的细胞毒性明显大于α-HBCD,γ-HBCD没有明显的细胞毒性。α-HBCD、β-HBCD对N2a细胞增殖的抑制作用呈时间-剂量效应关系(P<0.05),其半数抑制浓度(IC_(50))分别为60.07和10.52μmol/L,γ-HBCD的细胞毒性较小,镜下可见黑色絮状物,CCK-8法未能测定出其IC_(50);α-、β-HBCD会使细胞周期阻滞在G2/M期;染毒24 h后,RXRα、PPARγ、PXR及CYP3A11的mRNA及蛋白表达水平均呈现上升趋势(P<0.05);在N2a细胞内,α-HBCD染毒前后,RXRα与PPARγ、PXR之间始终存在交互作用关系。结论α-HBCD、β-HBCD对N2a细胞具有增殖抑制作用,细胞周期主要阻滞在G2/M期。α-HBCD、β-HBCD均可诱导3种细胞核受体RXRα、PPARγ和PXR的表达升高,PXR受体下游表达基因CYP3A11的表达也明显升高(P<0.05)。RXRα与PPARγ、PXR三个细胞核受体之间始终存在交互作用,但是受体间相互作用的分子机制有待深入研究。
Objective To investigate the effects of HBCDs on the proliferation of mouse neuroblastoma cell line N2a and the effects of three important nuclear receptors: retinal X receptor α (RXRα), peroxisome proliferator Effects of PPARγ, PXR Expression and Their Interactions. Methods N2a cells were treated with three concentrations of (±) α-HBCD, (±) β-HBCD and (±) γ-HBCD at different concentrations and Cell Count kit-8 On the cytotoxicity of N2a. Flow cytometry was used to detect the effect of HBCD on the cell cycle of N2a cells. RT-PCR and Western blotting were used to detect the expression of three nuclear receptors RXRα, PPARγ, PXR and the downstream target gene cytochrome P450 enzyme CYP3A11 mRNA and protein expression levels; co-immunoprecipitation analysis of RXRα, PXR, PPARγ receptor interactions. Results The cytotoxicity of β-HBCD to N2a was significantly greater than that of α-HBCD, and γ-HBCD had no obvious cytotoxicity. The inhibitory effects of α-HBCD and β-HBCD on the proliferation of N2a cells were time-dose-dependent (P <0.05), and the IC 50 values were 60.07 and 10.52μmol / L, respectively. Cytotoxicity was observed under microscope. Black floc was observed under microscope. IC 50 was not detected by CCK-8 method. Α-and β-HBCD blocked cell cycle in G2 / M phase. The mRNA and protein expression levels of RXRα, PPARγ, PXR and CYP3A11 all showed an upward trend (P <0.05). There was always an interaction between RXRα and PPARγ and PXR in N2a cells before and after α-HBCD treatment. Conclusion α-HBCD and β-HBCD can inhibit the proliferation of N2a cells. The cell cycle mainly arrests in G2 / M phase. The expression of RXRα, PPARγ and PXR in three nuclear receptors induced by α-HBCD and β-HBCD increased, and the expression of CYP3A11, a gene downstream of PXR, was also significantly increased (P <0.05). There is always an interaction between RXRα and PPARγ and PXR, but the molecular mechanism of the interaction between receptors needs further study.