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甲炔诺酮对斑马鱼胚胎下丘脑-垂体-生长轴基因表达的影响

作者:未知

  摘要 [目的] 研究甲炔诺酮对水生生物的生态毒理效应。
  [方法] 将受精后的斑马鱼胚胎分别暴露在含0、6、45和87 ng/L的甲炔诺酮水环境中144 h,研究甲炔诺酮对斑马鱼胚胎下丘脑-垂体-生长轴相关基因转录表达的影响。[结果] 甲炔诺酮在不同暴露时间点可以调节生长激素释放激素(ghrh)、胰岛素生长因子1(igf1)和胰岛素生长因子2b(igf2b)基因的转录表达,可以诱导生长激素(gh)基因的转录表达,但会抑制胰岛素生长因子1受体b(igf1rb)基因的转录表达。此外,甲炔诺酮暴露对生长激素受体(ghra、ghrb)、胰岛素生长因子2a(igf2a)和胰岛素生长因子1受体a(igf1ra)基因的转录表达没有显著影响。[结论] 甲炔诺酮能够改变斑马鱼早期胚胎发育阶段下丘脑-垂体-生长轴相关基因的转录表达水平,进而对斑马鱼的生长发育具有潜在的危险。
  关键词 孕激素;甲炔诺酮;下丘脑-垂体-生长轴;基因表达
  中图分类号 S912文献标识码 A
  文章编号 0517-6611(2019)10-0094-03
  Abstract [Objective]To study the ecotoxicological effect of norgestrel on aquatic organisms.[Method]Zebrafish embryos were exposed to 0,6,45 and 87 ng/L norgestrel for 144 h,and the effects of norgestrel on the transcriptional expression of the related genes with the hypothalamicpituitarygrowth axis in zebrafish embryos were studied.[Result]Norgestrel could mediate the transcriptional expression of growth hormonereleasing hormone (ghrh),insulinlike growth factor 1 (igf1) and insulinlike growth factor 2b (igf2b) genes at different exposure time points.Norgestrel exposure could induce the transcriptional expression of growth hormone (gh) gene,but it inhibited the transcriptional expression of insulinlike growth factor 1 receptor,beta (igf1rb) gene.In addition,norgestrel exposure had no significant effect on the transcriptional  expression of target genes associated with growth hormone receptor (ghra,ghrb), insulinlike growth factor 2a (igf2a) and insulinlike growth factor 1 receptor,alpha (igf1ra) genes.[Conclusion]Norgestrel could  affect the transcriptional expression levels of the related genes with hypothalamicpituitarygrowth axis,so it had potential risks on the growth of zebrafish.
  Key words Progestin;Norgestrel;Hypothalamicpituitarygrowth axis;Gene expression
  近年来,已有研究报道孕激素在水环境的污染分布特征,发现它们广泛存在于污水处理厂、养殖场和地表水等环境介質中,浓度范围从几纳克每升到几万纳克每升不等[1-2]。其中,甲炔诺酮作为主要的孕激素污染物之一,在地表水中的检出浓度为22 ng/L[3]。甲炔诺酮也残留在城市污水处理厂出水中,平均浓度为11 ng/L[4]。另外,在养猪场和奶牛场冲刷水中还检出相当高浓度的甲炔诺酮,浓度分别为10 800和6 460 ng/L[5-6]。甲炔诺酮在养猪场废水排放河流中也有检出,浓度高达465 ng/L[5]。目前报道的孕激素含量往往要比雌激素高出许多倍。然而,关于雌激素的预测无效应浓度有不少学者定为1 ng/L,即当水环境中雌激素含量大于1 ng/L时会对水生生物产生内分泌干扰效应[7]。因此,环境中高浓度甲炔诺酮的内分泌干扰效应应该引起高度重视,需要进一步研究。
  目前,已有研究报道了孕激素(如黄体酮、左炔诺孕酮、炔诺酮、孕二烯酮、屈螺酮、去氧孕烯、醋酸甲地孕酮和去氢孕酮)在ng/L水平上就能抑制鱼的生殖功能[8-14]。Liang等[15]研究表明低浓度的甲炔诺酮没有抑制斑马鱼的产卵量。但是,长期暴露于低浓度的甲炔诺酮能够干扰斑马鱼的性别分化,引起更多的雄鱼产生[16]。此外,甲炔诺酮还会干扰斑马鱼早期胚胎发育阶段下丘脑-垂体-性腺轴和下丘脑-垂体-甲状腺内分泌系统[17-18]。然而,目前关于甲炔诺酮对鱼类下丘脑-垂体-生长轴的影响尚未见报道。笔者将斑马鱼胚胎暴露于甲炔诺酮144 h,每天测定下丘脑-垂体-生长轴相关基因的转录表达水平,进而探讨甲炔诺酮对鱼类生长轴的潜在风险。   1 材料与方法
  1.1 实验动物
  成年斑马鱼购自广州市花鸟鱼虫市场,在实验室驯养至稳定后方可用于试验。将雌雄斑马鱼进行配对产卵,并将受精卵收集起来,用胚胎培养液冲洗干净。挑选发育正常且处于囊胚期(受精后2~4 h)的受精卵用于暴露试验。
  1.2 试验化合物 试验所用化合物为甲炔诺酮(norgestrel),纯度100%,用无水乙醇将其配制成浓度1 mg/mL的储备液,置于-20 ℃冰箱中保存备用。
  1.3 胚胎暴露试验设计
  甲炔诺酮浓度设置3个名义浓度,分别为5、50和100 ng/L,另设置溶剂对照组。甲炔诺酮处理组和溶剂对照组均含有0.001%的乙醇助溶剂。每个处理组设置4个平行,每个平行放入160个发育正常且处于囊胚期的胚胎。连续暴露144 h,在此期间每隔24 h换水1次。暴露期间,将胚胎放入人工气候箱中培养,温度保持在(26±1)℃,光暗周期比为14 h:10h。在暴露24、48、72、96、120和144 h时,每个平行随机挑选15个胚胎/幼体,保存于RNAlater中,用于此后的基因转录表达分析。
  1.4 基因转录表达分析
  根据Liang[17-18]的研究方法,对斑马鱼胚胎/幼体进行RNA提取、cDNA合成和实时荧光定量PCR。选取9个与下丘脑-垂体-生长轴相关的基因,检测了斑马鱼早期发育阶段生长激素释放激素(ghrh)、生长激素(gh)、生长激素受体(ghra和ghrb)、胰岛素生长因子(Igf1、Igf2a和Igf2b)以及胰岛素生长因子受体(Igf1ra和Igf1rb)基因的转录表达水平。采用β-actin、RpL13α和EF1-α这3个内参基因的平均值对目标基因进行归一化处理。参照Livak等[19]的2-ΔΔCt方法计算目标基因相对于参照因子(溶剂对照组的样品)的相对表达量,并以2为底数取对数值。
  1.5 暴露水溶液中甲炔诺酮的实测浓度
  为了测定甲炔诺酮在暴露水中的实际浓度,需要对溶剂对照组和各处理组水样进行化学分析,水样的采集和分析测定参照Liang[17-18]的方法。甲炔诺酮在3个浓度处理组的平均实测浓度为6、45和87 ng/L,而溶剂对照组未检测到甲炔诺酮[17-18]。以下试验所有暴露浓度数据都以它们的实测浓度表示。
  1.6 数据统计与分析
  试验数据均以平均值±标准差表示,采用SPSS 13.0统计软件进行数据分析,并用OriginPro 2016进行绘图。采用单因素方差分析方法(ANOVA)中的Tukey多重比较法检验不同浓度组各个基因的转录表达差异。
  2 结果与分析
  2.1 不同浓度甲炔诺酮暴露对斑马鱼早期胚胎发育过程中
  2.2 不同浓度甲炔诺酮暴露对斑马鱼早期胚胎发育过程中生长激素(gh)及其受体(ghra和ghrb)基因转录表达的影响
  2.3 不同浓度甲炔诺酮暴露对斑马鱼早期胚胎发育过程中胰岛素生长因子(Igf1、Igf2a和Igf2b)及其受体(Igf1ra和Igf1rb)基因转录表达的影响 从图3可以看出,6 ng/L甲炔诺酮暴露24 h提高了igf1基因的转录表达,而45 ng/L甲炔诺酮暴露72 h抑制了其转录表达水平。45和87 ng/L的甲炔诺酮分别暴露72和144 h降低了igf2b基因的转录表达水平,但暴露120 h提高了其转录水平。此外,6、45和87 ng/L的甲炔诺酮分别暴露72和144 h抑制了igf1rb基因的转录表达水平。然而,甲炔诺酮的暴露对igf2a和igf1ra基因的转录表达没有产生影响。
  3 讨论
  鱼类的生长发育主要与下丘脑-垂体-生长轴的调控有关,生长激素释放激素(growth hormonereleasing hormone,GHRH)、生长激素(growth hormone,GH)、生长激素受体(growth hormone receptor,GHR)、胰岛素生长因子(insulinlike growth factor,IGF)以及胰岛素生长因子受体(insulinlike growth factor receptor,IGFR)在鱼类的生长发育中起到非常重要的作用。其中,下丘脑分泌的GHRH的生理功能是調节垂体中GH的释放和合成,而GH是调控鱼类生长发育、繁殖和免疫的重要激素。GH通过与膜上的GHR结合,从而刺激IGF的合成与分泌,进而与IGFR结合来发挥其生物学效应。该研究结果表明,在斑马鱼早期胚胎发育阶段6 ng/L甲炔诺酮暴露能够改变下丘脑-垂体-生长轴相关的一些基因的转录水平。
  甲炔诺酮分别暴露24和144 h对ghrh基因的转录水平有促进作用,而甲炔诺酮分别暴露72和96 h则抑制了ghrh基因的转录水平,暗示了ghrh基因的转录变化是时间依赖模式,可能与发育阶段有关。关于甲炔诺酮这种时间依赖转录模式以前已有文献报道[17-18,20]。通常来说,成鱼的生长激素释放激素(GHRH)主要调节成年个体大脑中垂体生长激素(GH)的合成和分泌,因此ghrh基因的转录表达变化可能会改变GHRH的激素水平,从而调节GH的合成。该研究发现甲炔诺酮改变了gh基因的转录表达水平。
  生长激素(GH)调节着胰岛素生长因子(IGF)的合成和分泌。该研究发现甲炔诺酮暴露24 h显著提高了gh基因的转录水平,进而可能会干扰GH的合成,从而影响IGF的转录表达水平。该研究发现igf1、igf2b、igf1rb基因的转录表达水平在甲炔诺酮的处理下发生改变。
  综上所述,甲炔诺酮暴露能够干扰斑马鱼早期胚胎发育时期下丘脑-垂体-生长轴相关基因的转录表达,进而是否会对后期的生长发育产生影响则需要进一步研究。
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