三苯基锡与维甲酸X受体抑制剂UVI3003对爪蟾胚胎的致畸机制

发布时间：2018-08-19 14:56

【摘要】：维甲酸X受体(RetinoidXreceptor,RXR)是多种环境污染物和药物作用的重要靶点,三苯基锡(triphenyltin,TPT)是典型的RXR激动剂。有机锡被认为是人为引入海洋系统中的致毒效应最大的化学物质之一。然而,我们前期的研究却发现,TPT对热带爪蟾胚胎的致畸效应与RXR的激动剂9-顺式维甲酸(9-cis retinoic acid,9c-RA)、氟蓓萨罗汀(Fluorobexarotene,FBA)的截然不同,而与RXR高选择性抑制剂UVI3003的非常相似。因此,我们猜测在热带爪蟾胚胎中,TPT与UVI3003的致畸机制可能存在相似之处。本论文以热带爪蟾胚胎为实验模型,系统运用了基因组学、qRT-PCR定量、显微注射、整胚原位杂交、细胞转染等技术,比较研究了 TPT和UVI3003对热带爪蟾胚胎的致畸机制。首先,根据热带爪蟾胚胎的生长发育阶段特征,在NF 10-43时期划分出7个暴露窗口,分别进行UVI3003处理。UVI3003对不同发育时期的热带爪蟾胚胎引起的畸形表型和畸形程度不同,致畸能力在胚胎NF 10-25阶段较弱,NF 25-39时期明显增加,NF39-43时期又有所降低。UVI3003在热带爪蟾胚胎中引起的特征性畸形表型包括:小头、晶状体浑浊、泄殖腔异常、皮肤色素减少和窄鳍。UVI3003的qRT-PCR数据显示,与RXR相关的核受体基因在胚胎整个发育过程中受到的影响具有阶段差异性。但是,UVI3003在整个热带爪蟾胚胎暴露过程中都明显抑制了PPAγ基因的表达,同时上调了受PPARy负调控的基因mmp1。TPT也同样地对PPARγ和mmp1的表达值产生了相似的的影响。以上结果说明PPARγ基因在UVI3003致畸过程中具有重要作用。基因芯片结果显示,在温和作用窗口下,250、500、750μg/LUVI3003共同影响了rps15,serp2,fmr1,cyp2e1 lrrc9,ugtla 和LOC100490188七个基因的表达,主要影响的作用通路包括糖代谢、胰岛素信号通路和类固醇信号通路;在敏感作用模式下,18个基因被显著调控,UVI3003主要干扰了 PPAR信号通路、谷胱甘肽代谢和氮代谢。RXR-RAR被认为是维甲酸(Retinoic acid,RA)致畸的首要作用途径,但是在温和作用和敏感作用两种模式下都没有发现UVI3003对RA信号通路的影响。根据以上结果,我们推测PPAR信号通路更有可能是UVI3003对热带爪蟾胚胎致畸效应的潜在机制。进一步的核受体活性荧光素酶报告基因检测实验发现,在体外(Cos7细胞)、与爪蟾胚胎体内TPT与UVI3003都可以有效地激活爪蟾PPARγ;同时,UVI3003却对人类PPARy和小鼠PPARy基本没有活性。由此说明了激活PPARy是导致TPT与UVI3003在爪蟾胚胎体内引起相似畸形表型的潜在机制。本研究首次提出了 RXR高选择性抑制剂UVI3003同时是爪蟾特异的PPARy核受体激动剂的新观点,揭示了 TPT和UVI3003对爪蟾胚胎的致畸机制。为了研究PPARγ基因在TPT致畸过程中的功能作用,我们利用显微注射方法,在两细胞期热带爪蟾胚胎的单侧分别注射150 pgPPARγ mRNA和10 ng E2I2 Morpholino,从而过表达和敲落PPARγ基因。之后,将注射过的胚胎再次暴露在0、5、15、20 μgSn/LTPT溶液中。PPARγ过表达可以导致热带爪蟾胚胎的眼睛变小,在TPT暴露后此畸形表型得到了拯救;PPARγ基因敲落具有很强的致畸性,导致了热带爪蟾胚胎的多种畸形表型,这种致畸性与20 μgSn/LTPT暴露可以产生毒性叠加效应,导致胚胎死亡。PPP4Rγ的时空表达模式显示,在神经胚时期,PPARγ主要集中在神经嵴和神经管中表达。以上结果说明PPARγ基因下调的强致畸性在TPT致畸过程中具有重要贡献。以上结果在敏感基因PPARγ对TPT暴露的响应与胚胎眼睛畸形表型特征之间建立了联系。最后,为了研究RXR在维甲酸致畸过程中的的作用贡献,本文分别选取了RAR与RXR的选择性激动剂全反维甲酸(all-trans retinoic acid,at-RA)、氟蓓萨罗汀(FBA)以及二者的共同激动剂9-顺式维甲酸(9c-RA),利用基因芯片技术,比较解析三种激动剂对热带爪蟾胚胎基因表达谱的影响。由于三种激动剂引起的畸形表型高度相似,本文利用图像处理软件具体测量了每一种特征性表型,给出了定量的表型数据,辨别出了三种激动剂致畸效应的细微差异。基因芯片结果显示,RAR激动剂at-RA主要干扰了视黄醇代谢、糖酵解、淀粉和蔗糖代谢信号通路,RXR激动剂则产生了更加广泛的干扰,除了影响能量稳态方面的通路以外,还干扰了脂肪细胞因子信号通路、胰岛素耐受性、FoxO信号通路和PPAR信号通路。RXR是核受体家族中多个核受体的配体,这一特性开启了以下可能,RXR激动剂可以通过激活RXR影响到多种容许型二聚体下游的生物效应,如RXR-PPAR。因此,我们认为RXR激动剂的致畸作用途径不只是通过已知的RXR-RAR通路,RXR-PPAR在致畸过程中也起到了重要作用。综上所述,本文首次提出了 RXR高选择性抑制剂UVI3003同时是爪蟾特异的PPARγ核受体激动剂的新观点;揭示了激活PPARγ是导致TPT与UVI3003在爪蟾胚胎体内引起相似畸形表型的潜在机制;建立了敏感基因PPARγ对TPT暴露的响应与胚胎眼睛畸形表型特征之间的联系。同时,本文利用基因芯片技术筛选出了大量的维甲酸化合物的敏感作用基因,为建立RXR与RAR干扰物的筛选方法提供了分子指标数据。
[Abstract]:Retinoid X receptor (RXR) is an important target for a variety of environmental pollutants and drugs. Triphenyltin (TPT) is a typical RXR agonist. Organotin is considered to be one of the most toxic chemicals introduced into the marine system artificially. However, our previous studies have found that TPT has the greatest toxic effect on Xenopus embryos. The teratogenic effects of RXR agonists 9-cis retinoic acid (9c-RA), fluorobexarotine (FBA) are quite different from those of RXR highly selective inhibitors UVI3003. Therefore, we speculate that TPT and UVI3003 may have similar teratogenic mechanisms in Xenopus laevis embryos. The teratogenic mechanism of Xenopus laevis embryos induced by TPT and UVI 3003 was studied by genomics, qRT-PCR quantitative analysis, microinjection, whole embryo in situ hybridization and cell transfection. Firstly, according to the characteristics of Xenopus laevis embryos, seven exposure windows were divided in the period of NF 10-43. The teratogenic ability of UVI3003 was weaker in the stage of NF 10-25, increased significantly in the stage of NF 25-39, and decreased in the stage of NF 39-43. The characteristic phenotypes of malformation caused by UVI3003 in the embryos of Xenopus laevis were microcephaly and lens. The QRT-PCR data of UVI3003 showed that the effects of RXR-related nuclear receptor genes on the whole embryonic development were different in stages. However, UVI3003 significantly inhibited the expression of PPA-gamma gene and up-regulated the negative expression of PPARy during the whole embryonic exposure of Xenopus tropicalis. These results suggest that the PPAR gamma gene plays an important role in the teratogenic process of UVI3003. The results of gene chip analysis show that 250,500,750 ug/LUVI3003 together affect rps15, serp2, fmr1, CYP2E1 lrrc9, ugtla and LOC1004 under mild window. The expression of 901887 genes is mainly affected by glucose metabolism, insulin signaling pathway and steroid signaling pathway. Under the sensitive mode, 18 genes are significantly regulated. UVI 3003 mainly interferes with PPAR signaling pathway, glutathione metabolism and nitrogen metabolism. RXR-RAR is considered to be the first teratogenic pathway of retinoic acid (RA). According to the above results, we speculate that the PPAR signaling pathway is more likely to be the potential mechanism of teratogenic effect of UVI3003 on Xenopus tropicalis embryos. It was found that both TPT and UVI3003 could effectively activate Xenopus laevis PPARy in vitro (Cos7 cells) and Xenopus laevis embryos, while UVI3003 had little activity on human PPARy and mouse PPARy. This indicated that activation of PPARy was a potential mechanism leading to similar abnormal phenotypes of TPT and UVI3003 in Xenopus laevis embryos. The new view that XR highly selective inhibitor UVI3003 is a Xenopus-specific PPARy nuclear receptor agonist at the same time reveals the teratogenic mechanism of TPT and UVI3003 on Xenopus laevis embryos. In order to study the function of PPARgamma gene in the teratogenic process of TPT, we injected 150 pgPPA into Xenopus laevis embryos by microinjection. Rgamma mRNA and 10NG E2I2 Morpholino overexpressed and knocked out the PPAR gamma gene. The injected embryos were then re-exposed to 0,5,15,20 UG Sn/LTPT solution. Several abnormal phenotypes of Xenopus tropicalis embryos were identified. The teratogenicity and exposure to 20 UG Sn/LTPT produced a toxic superposition effect leading to embryo death. The expression pattern of PPP4R gamma in the neural crest and canal during the embryonic period showed that the expression of PPAR gamma was mainly in the neural crest and canal. These results have established a link between the response of the sensitive gene PPARgamma to TPT exposure and the phenotypic characteristics of embryonic eye malformations. Finally, in order to study the role of RXR in retinoic acid teratogenesis, all-trans retinoic acid (at-trans retinoic acid) was selected as the selective agonist of RAR and RXR, respectively. RA, FBA and their co-agonists 9-cis-retinoic acid (9c-RA) were used to compare and analyze the effects of the three agonists on gene expression profiles of Xenopus laevis embryos by gene chip technique. The results of gene chip analysis showed that RAR agonist at-RA mainly interfered with retinol metabolism, glycolysis, starch and sucrose metabolism signaling pathways, while RXR agonists interfered with a wider range of energy homeostasis pathways. It also interferes with adipocytokine signaling pathways, insulin tolerance, FoxO signaling pathways, and PPAR signaling pathways. RXR is the ligand of multiple nuclear receptors in the nuclear receptor family. This characteristic opens the possibility that RXR agonists can affect the downstream biological effects of multiple allowable dimers, such as RXR-PPAR, by activating RXR. RXR-PPAR plays an important role in the teratogenic process as well as through the known RXR-RAR pathway. In conclusion, a new viewpoint that the highly selective inhibitor of RXR, UVI3003, is also a Xenopus-specific PPAR gamma receptor agonist, has been proposed for the first time in this paper. The potential mechanism of Xenopus laevis embryos to induce similar phenotypes was established, and the relationship between the response of sensitive gene PPARgamma to TPT exposure and the phenotypic characteristics of embryonic eye malformation was established. Molecular index data are provided.
【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：X174

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