本文選題：T-2毒素 + 轉(zhuǎn)錄因子��； 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：T-2毒素在我國糧食作物和飼料中廣泛存在,具有毒性強(qiáng)、脫毒困難等特點(diǎn)。動物在長期攝入低劑量T-2毒素污染的谷物或飼料后就會引起中毒,生長發(fā)育遲緩,體重明顯下降,給畜牧業(yè)生產(chǎn)帶來巨大的經(jīng)濟(jì)損失。炎性因子的大量表達(dá)和生長激素(Growth hormone,GH)的缺乏是導(dǎo)致生長抑制的重要原因�；蚪M學(xué)的結(jié)果表明,10、40 nM的T-2毒素顯著下調(diào)GH表達(dá)的同時也顯著下調(diào)轉(zhuǎn)錄因子AKNA(AT-hook transcription factor)基因表達(dá)(分別下調(diào)25.85和52.15倍),且顯著上調(diào)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)基因的表達(dá),但是目前關(guān)于T-2毒素誘導(dǎo)GH下調(diào)的分子機(jī)制尚不清楚。轉(zhuǎn)錄因子AKNA與炎性因子的表達(dá)密切相關(guān),當(dāng)AKNA基因被敲除時,可導(dǎo)致新生小鼠的死亡和中性粒細(xì)胞介導(dǎo)的炎癥反應(yīng),引起IL-1β和干擾素-γ(Interferon-γ,IFN-γ)基因表達(dá)量增加,但是AKNA如何調(diào)控炎性細(xì)胞因子表達(dá)的目前并不清楚。因此推測,轉(zhuǎn)錄因子AKNA可能是T-2毒素誘導(dǎo)的GH下調(diào)的一個關(guān)鍵調(diào)控因子,T-2毒素通過抑制AKNA表達(dá)導(dǎo)致炎性反應(yīng)以及GH合成和分泌降低。我們以轉(zhuǎn)錄因子AKNA為切入點(diǎn),深入闡釋調(diào)控轉(zhuǎn)錄因子AKNA表達(dá)的分子機(jī)制以及AKNA在T-2毒素介導(dǎo)的炎性反應(yīng)和GH下調(diào)中的作用,確定毒素作用的關(guān)鍵靶點(diǎn)和信號通路,為保障動物健康,開發(fā)出高效、安全的新型拮抗劑提供科學(xué)依據(jù),并為全面評價T-2毒素的風(fēng)險(xiǎn)奠定基礎(chǔ)。分別用濃度為10 nM和40 nM的T-2毒素處理GH3細(xì)胞0.5、1、2、4、8和12 h,用濃度為5 nM、10 nM、20 nM、40 nM和80 nM的T-2毒素處理GH3細(xì)胞12 h,通過qRT-PCR檢測AKNA的mRNA表達(dá)水平與T-2毒素間的時效關(guān)系與量效關(guān)系,結(jié)果表明T-2毒素可呈時間依賴型和劑量依賴型的下調(diào)轉(zhuǎn)錄因子AKNA的表達(dá)。用不同的信號通路抑制劑預(yù)處理細(xì)胞后,再加入T-2毒素,以篩選T-2毒素作用下參與調(diào)控轉(zhuǎn)錄因子AKNA的信號通路,結(jié)果表明PKA/CREB、NF-κB和MAPK/p38信號通路抑制劑能顯著逆轉(zhuǎn)T-2毒素所致的AKNA表達(dá)的下調(diào)。通過生物信息學(xué)分析發(fā)現(xiàn)AKNA啟動子區(qū)含有PKA/CREB和NF-κB信號通路中關(guān)鍵蛋白CREB和p65基因上的結(jié)合位點(diǎn)。將構(gòu)建成功的CREB和p65過表達(dá)載體轉(zhuǎn)染到GH3細(xì)胞,結(jié)果顯示過表達(dá)CREB和p65后轉(zhuǎn)錄因子AKNA表達(dá)水平顯著下調(diào),表明CREB和p65可負(fù)調(diào)控AKNA的表達(dá),PKA/CREB和NF-κB信號通路可能是調(diào)控AKNA表達(dá)的關(guān)鍵信號通路。用40 nM的T-2毒素分別孵育GH3細(xì)胞0.5、1、2、4、8和12 h,采用間接免疫熒光雙標(biāo)技術(shù)結(jié)合激光共聚焦顯微鏡觀測CREB和p65的磷酸化入核過程和AKNA在細(xì)胞中的位置。未加毒素處理時,磷酸化CREB主要位于細(xì)胞核,磷酸化p65和AKNA主要位于細(xì)胞質(zhì);T-2毒素孵育后細(xì)胞核中的磷酸化CREB和磷酸化p65明顯增加,T-2毒素處理1 h后細(xì)胞核中的磷酸化CREB增加最明顯,T-2毒素處理8 h后細(xì)胞核中的磷酸化p65增加最明顯;轉(zhuǎn)錄因子AKNA在T-2毒素處理2 h后開始進(jìn)入細(xì)胞核,8 h后入核最明顯。由此可知,T-2毒素促使CREB和p65蛋白磷酸化并進(jìn)入細(xì)胞核,從而激活PKA/CREB和NF-κB/p65信號通路,調(diào)控下游基因的表達(dá);在大鼠細(xì)胞中轉(zhuǎn)錄因子AKNA主要位于細(xì)胞質(zhì),T-2毒素可促使AKNA進(jìn)入細(xì)胞核發(fā)揮毒理效應(yīng)。為了確定CREB和p65調(diào)控AKNA的作用方式,通過雙熒光素酶報(bào)告基因檢測發(fā)現(xiàn)CREB和p65可極顯著下調(diào)AKNA啟動子區(qū)域的轉(zhuǎn)錄活性。通過染色質(zhì)免疫共沉淀結(jié)合熒光定量PCR分析發(fā)現(xiàn),CREB蛋白和p65蛋白可分別與AKNA基因近端啟動子(-1862 bp和-1476 bp)結(jié)合,T-2毒素可顯著增加CREB和p65與AKNA啟動子區(qū)域的結(jié)合活性。由此可知,磷酸化CREB和磷酸化p65蛋白可直接與AKNA啟動子結(jié)合從而抑制AKNA的轉(zhuǎn)錄表達(dá),T-2毒素通過促進(jìn)磷酸化CREB和磷酸化p65蛋白與AKNA啟動子結(jié)合從而使AKNA表達(dá)下調(diào)。通過構(gòu)建pSicoR-AKNA干擾載體并轉(zhuǎn)染到GH3細(xì)胞抑制AKNA的表達(dá),確定轉(zhuǎn)錄因子AKNA下游調(diào)控因子及其在生長抑制中的作用。抑制轉(zhuǎn)錄因子AKNA表達(dá)后加毒素處理,與僅加毒素處理組相比炎性因子IL-1β、IL-6、IL-11、TNF-α和MMP-9基因表達(dá)量降低,說明AKNA可正調(diào)控炎性細(xì)胞因子和MMP-9的表達(dá),在T-2毒素介導(dǎo)的炎性因子和MMP-9上調(diào)表達(dá)中,除了AKNA調(diào)控炎性因子表達(dá)之外,還有其他的信號通路參與T-2毒素誘導(dǎo)的炎性因子的上調(diào)表達(dá)。抑制轉(zhuǎn)錄因子AKNA表達(dá)后還發(fā)現(xiàn)GH的表達(dá)顯著下調(diào),說明AKNA是介導(dǎo)GH下調(diào)的一個重要調(diào)控因子,在T-2毒素誘導(dǎo)GH下調(diào)的生長抑制中具有重要作用。轉(zhuǎn)錄因子AKNA很可能是T-2毒素誘導(dǎo)生長抑制的一個重要的新的靶標(biāo)。綜上所述:本課題以轉(zhuǎn)錄因子AKNA為靶標(biāo)研究T-2毒素介導(dǎo)的生長抑制毒性作用機(jī)理,系統(tǒng)的闡釋了調(diào)控AKNA表達(dá)的分子機(jī)制以及AKNA在T-2毒素誘導(dǎo)的GH下調(diào)和炎性細(xì)胞因子大量表達(dá)中的作用。T-2毒素可以誘導(dǎo)PKA/CREB和NF-κB/p65信號通路中關(guān)鍵蛋白CREB和p65表達(dá)增加,增加CREB和p65蛋白磷酸化水平,促使磷酸化p65進(jìn)入細(xì)胞核以及核內(nèi)的磷酸化CREB增加從而激活PKA/CREB和NF-κB/p65信號通路,核內(nèi)磷酸化的CREB和p65通過直接與AKNA啟動子結(jié)合抑制AKNA的轉(zhuǎn)錄活性,從而使AKNA的基因表達(dá)顯著下調(diào)。在大鼠細(xì)胞中轉(zhuǎn)錄因子AKNA主要位于細(xì)胞質(zhì),T-2毒素作用后進(jìn)入細(xì)胞核發(fā)揮毒理效應(yīng)。抑制轉(zhuǎn)錄因子AKNA表達(dá)后GH和炎性因子的表達(dá)顯著下調(diào),表明AKNA參與調(diào)控GH和炎性因子的表達(dá),為T-2毒素介導(dǎo)的GH缺乏癥提供一個新的下游分子靶標(biāo),且揭示了AKNA的分子調(diào)控機(jī)制,為以后AKNA分子調(diào)控方面的研究提供了重要參考,為T-2毒素的毒理機(jī)制和毒素防控提供了重要依據(jù)。
[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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S859.8

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