转录因子AKNA在T-2毒素诱导的生长抑制毒性中的作用研究

发布时间：2018-05-14 12:37

本文选题：T-2毒素 + 转录因子　；参考：《华中农业大学》2017年硕士论文

【摘要】：T-2毒素在我国粮食作物和饲料中广泛存在,具有毒性强、脱毒困难等特点。动物在长期摄入低剂量T-2毒素污染的谷物或饲料后就会引起中毒,生长发育迟缓,体重明显下降,给畜牧业生产带来巨大的经济损失。炎性因子的大量表达和生长激素(Growth hormone,GH)的缺乏是导致生长抑制的重要原因。基因组学的结果表明,10、40 nM的T-2毒素显著下调GH表达的同时也显著下调转录因子AKNA(AT-hook transcription factor)基因表达(分别下调25.85和52.15倍),且显著上调白介素-6(Interleukin-6,IL-6)、白介素-11(Interleukin-11,IL-11)和白介素-1β(Interleukin-1β,IL-1β)基因的表达,但是目前关于T-2毒素诱导GH下调的分子机制尚不清楚。转录因子AKNA与炎性因子的表达密切相关,当AKNA基因被敲除时,可导致新生小鼠的死亡和中性粒细胞介导的炎症反应,引起IL-1β和干扰素-γ(Interferon-γ,IFN-γ)基因表达量增加,但是AKNA如何调控炎性细胞因子表达的目前并不清楚。因此推测,转录因子AKNA可能是T-2毒素诱导的GH下调的一个关键调控因子,T-2毒素通过抑制AKNA表达导致炎性反应以及GH合成和分泌降低。我们以转录因子AKNA为切入点,深入阐释调控转录因子AKNA表达的分子机制以及AKNA在T-2毒素介导的炎性反应和GH下调中的作用,确定毒素作用的关键靶点和信号通路,为保障动物健康,开发出高效、安全的新型拮抗剂提供科学依据,并为全面评价T-2毒素的风险奠定基础。分别用浓度为10 nM和40 nM的T-2毒素处理GH3细胞0.5、1、2、4、8和12 h,用浓度为5 nM、10 nM、20 nM、40 nM和80 nM的T-2毒素处理GH3细胞12 h,通过qRT-PCR检测AKNA的mRNA表达水平与T-2毒素间的时效关系与量效关系,结果表明T-2毒素可呈时间依赖型和剂量依赖型的下调转录因子AKNA的表达。用不同的信号通路抑制剂预处理细胞后,再加入T-2毒素,以筛选T-2毒素作用下参与调控转录因子AKNA的信号通路,结果表明PKA/CREB、NF-κB和MAPK/p38信号通路抑制剂能显著逆转T-2毒素所致的AKNA表达的下调。通过生物信息学分析发现AKNA启动子区含有PKA/CREB和NF-κB信号通路中关键蛋白CREB和p65基因上的结合位点。将构建成功的CREB和p65过表达载体转染到GH3细胞,结果显示过表达CREB和p65后转录因子AKNA表达水平显著下调,表明CREB和p65可负调控AKNA的表达,PKA/CREB和NF-κB信号通路可能是调控AKNA表达的关键信号通路。用40 nM的T-2毒素分别孵育GH3细胞0.5、1、2、4、8和12 h,采用间接免疫荧光双标技术结合激光共聚焦显微镜观测CREB和p65的磷酸化入核过程和AKNA在细胞中的位置。未加毒素处理时,磷酸化CREB主要位于细胞核,磷酸化p65和AKNA主要位于细胞质;T-2毒素孵育后细胞核中的磷酸化CREB和磷酸化p65明显增加,T-2毒素处理1 h后细胞核中的磷酸化CREB增加最明显,T-2毒素处理8 h后细胞核中的磷酸化p65增加最明显;转录因子AKNA在T-2毒素处理2 h后开始进入细胞核,8 h后入核最明显。由此可知,T-2毒素促使CREB和p65蛋白磷酸化并进入细胞核,从而激活PKA/CREB和NF-κB/p65信号通路,调控下游基因的表达;在大鼠细胞中转录因子AKNA主要位于细胞质,T-2毒素可促使AKNA进入细胞核发挥毒理效应。为了确定CREB和p65调控AKNA的作用方式,通过双荧光素酶报告基因检测发现CREB和p65可极显著下调AKNA启动子区域的转录活性。通过染色质免疫共沉淀结合荧光定量PCR分析发现,CREB蛋白和p65蛋白可分别与AKNA基因近端启动子(-1862 bp和-1476 bp)结合,T-2毒素可显著增加CREB和p65与AKNA启动子区域的结合活性。由此可知,磷酸化CREB和磷酸化p65蛋白可直接与AKNA启动子结合从而抑制AKNA的转录表达,T-2毒素通过促进磷酸化CREB和磷酸化p65蛋白与AKNA启动子结合从而使AKNA表达下调。通过构建pSicoR-AKNA干扰载体并转染到GH3细胞抑制AKNA的表达,确定转录因子AKNA下游调控因子及其在生长抑制中的作用。抑制转录因子AKNA表达后加毒素处理,与仅加毒素处理组相比炎性因子IL-1β、IL-6、IL-11、TNF-α和MMP-9基因表达量降低,说明AKNA可正调控炎性细胞因子和MMP-9的表达,在T-2毒素介导的炎性因子和MMP-9上调表达中,除了AKNA调控炎性因子表达之外,还有其他的信号通路参与T-2毒素诱导的炎性因子的上调表达。抑制转录因子AKNA表达后还发现GH的表达显著下调,说明AKNA是介导GH下调的一个重要调控因子,在T-2毒素诱导GH下调的生长抑制中具有重要作用。转录因子AKNA很可能是T-2毒素诱导生长抑制的一个重要的新的靶标。综上所述:本课题以转录因子AKNA为靶标研究T-2毒素介导的生长抑制毒性作用机理,系统的阐释了调控AKNA表达的分子机制以及AKNA在T-2毒素诱导的GH下调和炎性细胞因子大量表达中的作用。T-2毒素可以诱导PKA/CREB和NF-κB/p65信号通路中关键蛋白CREB和p65表达增加,增加CREB和p65蛋白磷酸化水平,促使磷酸化p65进入细胞核以及核内的磷酸化CREB增加从而激活PKA/CREB和NF-κB/p65信号通路,核内磷酸化的CREB和p65通过直接与AKNA启动子结合抑制AKNA的转录活性,从而使AKNA的基因表达显著下调。在大鼠细胞中转录因子AKNA主要位于细胞质,T-2毒素作用后进入细胞核发挥毒理效应。抑制转录因子AKNA表达后GH和炎性因子的表达显著下调,表明AKNA参与调控GH和炎性因子的表达,为T-2毒素介导的GH缺乏症提供一个新的下游分子靶标,且揭示了AKNA的分子调控机制,为以后AKNA分子调控方面的研究提供了重要参考,为T-2毒素的毒理机制和毒素防控提供了重要依据。
[Abstract]:T-2 toxin is widely existed in grain crops and forage in our country. It has the characteristics of strong toxicity and detoxification. The animals will be poisoned by the long intake of low dose T-2 toxin contaminated grain or feed, and the growth retardation and weight decrease obviously. It brings huge economic loss to the production of animal husbandry. The large amount of expression and growth of inflammatory factors The lack of Growth hormone (GH) is an important cause of growth inhibition. The results of genomics showed that the T-2 toxin of 10,40 nM significantly lowered the expression of GH and down regulated the expression of the transcriptional factor AKNA (AT-hook transcription factor) (25.85 and 52.15 times respectively), and significantly up-regulated the interleukin -6. The expression of interleukin -11 (Interleukin-11, IL-11) and interleukin -1 beta (Interleukin-1 beta, IL-1 beta) genes, but the molecular mechanism of the T-2 toxin induced down regulation of GH is still unclear. The transcription factor AKNA is closely related to the expression of inflammatory factors. When the AKNA gene is knocked out, it can lead to the death of newborn mice and neutrophil mediated inflammation. The expression of IL-1 beta and interferon gamma (Interferon- gamma, IFN- gamma) gene expression increases, but it is not clear how AKNA regulates the expression of inflammatory cytokines. Therefore, it is presumed that the transcription factor AKNA may be a key regulator of the downregulation of GH induced by T-2 toxin, and T-2 toxin can lead to inflammatory reaction and GH by inhibiting AKNA expression. The synthesis and secretion decrease. We use the transcription factor AKNA as the breakthrough point to explain the molecular mechanism of the transcription factor AKNA expression and the role of AKNA in the T-2 toxin mediated inflammatory response and GH downregulation, to determine the key targets and signal pathways for the effect of the toxin, in order to protect the animal health and develop efficient and safe new antagonists. Scientific basis, and lay the foundation for comprehensive evaluation of the risk of T-2 toxin. GH3 cells 0.5,1,2,4,8 and 12 h were treated with T-2 toxin with a concentration of 10 nM and 40 nM respectively. The concentrations of 5 nM, 10 nM, 20 nM, 40 nM and 80 nM were treated. The results showed that T-2 toxin could be time dependent and dose-dependent down down transcription factor AKNA expression. After pretreating cells with different signal pathway inhibitors, T-2 toxin was added to screen signaling pathways involved in the regulation of transcription factor AKNA under the action of T-2 toxin. The results showed that PKA/CREB, NF- kappa B and MAPK/p38 signaling pathways were suppressed. The agent can significantly reverse the downregulation of AKNA expression caused by T-2 toxin. Through bioinformatics analysis, it is found that the AKNA promoter region contains the binding site of the key protein CREB and p65 gene in the PKA/CREB and NF- kappa B signaling pathway. The successful CREB and p65 overexpressed vectors are constructed to the GH3 cells. The results show that the expression CREB and the post transcription factor transcription factor are expressed. The expression level of KNA was significantly downregulated, indicating that CREB and p65 can negatively regulate the expression of AKNA. PKA/CREB and NF- kappa B signaling pathways may be the key signaling pathways regulating AKNA expression. GH3 cell 0.5,1,2,4,8 and 12 are incubated with 40 nM T-2 toxin, respectively, and indirect immunofluorescence double labeling technique is used to observe the phosphoric acid and phosphoric acid by laser confocal microscopy. The nucleation process and the location of AKNA in the cell. Phosphorylated CREB is mainly located in the nucleus, phosphorylated p65 and AKNA are mainly located in the cytoplasm; the phosphorylated CREB and phosphorylated p65 in the nucleus of the T-2 toxin increased obviously, and the phosphorylation CREB increased most obviously after the T-2 toxin treatment 1 h, and the T-2 toxin treated 8 h. The phosphorylation of p65 in the nucleus was increased most obviously; the transcription factor AKNA began to enter the nucleus after the treatment of T-2 toxin 2 h, and the nucleus was most obvious after 8 h. Thus, T-2 toxin stimulated CREB and p65 protein phosphorylation and entered the nucleus, thus activating PKA/CREB and NF- kappa B/p65 signaling pathway, regulating the expression of downstream genes, and transferring in rat cells. The recording factor AKNA is mainly located in the cytoplasm, and T-2 toxin can induce AKNA to enter the nucleus to play a toxic effect. In order to determine the way of CREB and p65 to regulate the action of AKNA, the transcriptional activity of CREB and p65 can be significantly down regulated by the double luciferase reporter gene detection. The chromatin immunoprecipitation combined with fluorescence quantitative PCR through the chromatin immunoprecipitation. It was found that CREB protein and p65 protein can bind to the proximal promoter of AKNA gene (-1862 BP and -1476 BP), and T-2 toxin can significantly increase the binding activity of CREB and p65 to AKNA promoter region. Promote the combination of phosphorylated CREB and phosphorylated p65 protein with AKNA promoter to reduce the expression of AKNA. By constructing pSicoR-AKNA interference carrier and transfecting GH3 cells to inhibit the expression of AKNA, the downstream regulatory factor of the transcription factor AKNA and its role in growth inhibition are determined. Compared with the inflammatory factors IL-1 beta, IL-6, IL-11, TNF- A and MMP-9 gene expression, AKNA can regulate the expression of inflammatory cytokines and MMP-9. In addition to T-2 toxin mediated inflammatory factors and MMP-9 up-regulated expression, there are other signaling pathways involved in T-2 toxin induced inflammation in addition to AKNA regulation of inflammatory factors. The up-regulated expression of factors. After the inhibition of the expression of the transcription factor AKNA, the expression of GH was significantly downregulated, suggesting that AKNA is an important regulator of the down regulation of GH, and it plays an important role in the growth inhibition of T-2 toxin induced down regulation of GH. The transcription factor AKNA is likely to be an important new target for the growth inhibition of T-2 toxin. This topic studies the mechanism of the inhibitory toxicity of T-2 toxin mediated by transcription factor AKNA, and systematically explains the molecular mechanism of regulating AKNA expression and the role of AKNA in T-2 toxin induced GH down regulation and the large number of inflammatory cytokines,.T-2 toxin can induce key proteins in PKA/CREB and NF- kappa B/p65 signaling pathway The expression of CREB and p65 increased, increasing the phosphorylation level of CREB and p65 protein, prompting the phosphorylated p65 to enter the nucleus and the phosphorylated CREB in the nucleus to activate the PKA/CREB and NF- kappa B/p65 signaling pathway. The CREB and p65 of phosphorylation in the nucleus could inhibit the transcriptional activity by binding directly with the AKNA promoter, thus making the gene expression significantly lower. In rat cells, the transcription factor AKNA is mainly located in the cytoplasm and T-2 toxin acts into the nucleus to play a toxic effect. The expression of GH and inflammatory factors is significantly downregulated after the inhibition of the transcription factor AKNA expression, indicating that AKNA participates in the regulation of the expression of GH and inflammatory factors and provides a new downstream molecular target for the GH deficiency mediated by T-2 toxin. In addition, the molecular regulation mechanism of AKNA has been revealed, which provides important reference for the study of AKNA molecular regulation in the future, and provides an important basis for the toxicological mechanism of T-2 toxin and the prevention and control of toxins.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.8

【参考文献】