PFOS对BV-2细胞的炎性损伤及机制研究
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国家自然科学基金(81202230,81473012);南京医科大学科技发展基金(2011NJMU275);江苏省高校优势学科建设工程资助项目(CX09B-264Z);临床医学科技专项(BL2014088)


Inflammatory damage of BV-2 cells induced by PFOS and its mechanism
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    摘要:

    目的:探讨环境内分泌干扰物全氟辛磺酸(perfluorooctane suifonate,PFOS)对小鼠小胶质瘤细胞BV-2的损伤作用及其炎性反应。方法:利用体外培养的小胶质细胞,给予不同浓度和作用时间的PFOS进行染毒,通过MTT法检测BV-2细胞活力;实时定量PCR的方法观察诱导型一氧化氮合酶(inducible nitric oxide synthesis,iNOS)、白介素(interleukin,IL)-6 和肿瘤坏死因子(tumor necrosis factor,TNF)-α mRNA的表达。ELISA法检测炎性因子IL-6,免疫蛋白印迹法检测核转录因子kappa B(nuclear factor-kappa B,NF-κB)等信号蛋白表达情况。结果:不同浓度PFOS对BV-2细胞染毒12、24 h后,细胞活力下降,引起明显的细胞损伤。ELISA结果显示, PFOS作用BV-2 24 h后,细胞炎性因子IL-6分泌增加。此外,PFOS还可引起iNOS、IL-6基因表达上调,而TNF-α表达有下降趋势。PFOS与细胞孵育后,炎性通路NF-κB明显激活,表现为磷酸化程度升高。且随染毒浓度的增加,p-NF-κB的表达有上升趋势;此外,随染毒时间的增加,p-NF-κB的表达亦有上升趋势。结论:PFOS可引起BV-2细胞活力降低,激活NF-κB通路,促进炎性因子的释放,该途径可为阐明PFOS引起的神经系统损伤的分子机制提供理论依据。

    Abstract:

    Objective: To investigate the effect of environmental endocrine disruptor perfluorooctane suifonate(PFOS) on the cell damage and expression of inflammatory factors in mouse BV-2 microglial cells. Methods: The microglial cells were cultured by different concentrations of PFOS with various periods in vitro, and the activity of BV-2 cells was detected by using MTT method. Real-time PCR assay was performed to detect the mRNA expression of the inflammatory factors, including inducible nitric oxide synthesis(iNOS), tumor necrosis factor(TNF)-α and interleukin(IL)-6. At protein level, ELISA was used to analyze the excretion of IL-6 in BV-2 cells mediated by PFOS. Moreover, the signal pathway, especially the nuclear factor-kappa B(NF-κB), was evaluated by Western blot. Results: The BV-2 cells were incubated with varied doses of PFOS for 12 or 24 h, and then the cell viability was significantly decreased, which led to significant cell damage. ELISA data showed a significant incretion of the cytokine factor IL-6 after BV-2 cells were incubated with PFOS for 24 h. PFOS markedly up-regulated iNOS and IL-6 mRNA expression, while TNF-α was down-regulated. Therefore, we discovered that PFOS substantially activated and phosphorylated the NF-κB pathway in a dose dependent manner. In addition, with the increase of exposure time, the expression of p-NF-κB was also increased. Conclusion: Our results showed that PFOS markedly decreased the BV-2 cell viability, activated the NF-κB pathway, and excreted the cytokines. These results taken together might provide a new molecular mechanism supporting the PFOS-induced neural damage.

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刘艳青,宋 词,王 军. PFOS对BV-2细胞的炎性损伤及机制研究[J].南京医科大学学报(自然科学版),2017,(6):681-685

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  • 收稿日期:2016-07-11
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  • 在线发布日期: 2017-06-30
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