本文選題：MC-LR + GT1-7�。� 參考：《南京大學》2017年碩士論文

【摘要】：微囊藻毒素(Microcystins,MCs)是淡水藍藻產(chǎn)生的一類由七個氨基酸組成的天然毒素,具有強烈的肝毒性、神經(jīng)毒性、腎毒性和胃腸道毒性[1-3]。本課題組率先發(fā)現(xiàn)MCs具有雄性生殖毒性。我們在前期研究中,分別通過急性和慢性實驗證明了MC-LR(水體中分布最廣、毒性最強的一種MCs異構(gòu)體)染毒雄性大鼠后,引起機體睪酮水平明顯下降,其下降幅度為60%～80%[4]。在雄性動物體內(nèi),睪丸間質(zhì)細胞是合成睪酮的重要場所。而體內(nèi)體外實驗均表明,MC-LR不能進入間質(zhì)細胞,對間質(zhì)細胞沒有明顯的損傷作用[5]。間質(zhì)細胞合成睪酮受下丘腦-腺垂體-睪丸軸的調(diào)控。下丘腦GnRH神經(jīng)元分泌的GnRH間接調(diào)控間質(zhì)細胞合成睪酮。前期結(jié)果表明,MC-LR能夠有效進入下丘腦組織,下調(diào)GnRH的量[6]。已知,GnRH由下丘腦中GnRH神經(jīng)元分泌。由于GnRH神經(jīng)元數(shù)量少,在下丘腦分布零散,我們使用GT1-7細胞進行后續(xù)的機制研究。GT1-7細胞能穩(wěn)定的分泌GnRH,是研究GnRH神經(jīng)元的理想細胞株[7-8]。本研究探討MC-LR進入GT1-7細胞后,抑制GnRH合成的分子機制。全文分為三部分:第一部分MC-LR對GT1-7細胞miRNA表達譜的影響一、目的探究MC-LR對GT1-7細胞miRNA表達譜的影響,篩選與GnRH合成相關(guān)的miRNA。二、方法1.GT1-7細胞均分10組進行各項指標的檢測,即Control、1nM、10nM、100nM、500nM、1μM、5μM、10μM、50μM、1000μM MC-LR組。2.光鏡下觀察細胞形態(tài),采用CCK-8法,檢測MC-LR對GC-1細胞形態(tài),活力和存活率的影響,確認GT1-7細胞最佳的染毒濃度和時間,為進行miRNA芯片篩選選擇最佳染毒條件。3.采用miRNA雜交芯片法,篩選出MC-LR染毒GT1-7細胞后,細胞中表達變化的miRNA。采用qPCR實驗技術(shù)驗證芯片的可靠性。4.miR-329-3p靶基因的預測和驗證:采用miRanda、Targetscan及microRNA.org等生物信息學軟件預測差異變化miRNA的靶基因,借此篩選出與GnRH合成相關(guān)的miRNA。分別構(gòu)建含Prkarla、Prkacb與miR-329-3p互補配對堿基序列的熒光素酶報告基因重組質(zhì)粒,采用脂質(zhì)體轉(zhuǎn)染外源性miR-329-3p或negative control和重組質(zhì)粒或空載質(zhì)粒進入293T細胞,驗證miR-329-3p與Prkarla、Prkacb之間的作用關(guān)系。5.使用Promega試劑盒檢測MC-LR染毒后GT1-7細胞中PKA的酶活性是否改變。三、結(jié)果1.采用CCK-8法檢測細胞活力發(fā)現(xiàn):隨著MC-LR染毒濃度和染毒時間的增加,細胞活性呈現(xiàn)下降趨勢。100nM-100μM組染毒48h,GT1-7細胞活力顯著降低。光鏡下觀察細胞的形態(tài)發(fā)現(xiàn):0-1000 nM組細胞形態(tài)未明顯改變,10 μM和100 μM組細胞呈圓形并懸浮于培養(yǎng)基中。2.MC-LR對GT1-7細胞miRNA表達譜的影響:500 nM MC-LR染毒GT1-7細胞48 h,通過對比GT1-7細胞染毒前后miRNA的變化,發(fā)現(xiàn)101種miRNA發(fā)生明顯改變,其中42種顯著上調(diào),59種顯著下調(diào)。其中,上調(diào)最明顯的是miR-544-3p,上調(diào)了 10.67倍。下調(diào)最明顯的是miR-329-3p,下調(diào)了 9.5倍。qPCR證實表達差異的miRNAs結(jié)果與芯片一致。3.通過綜合比對不同數(shù)據(jù)庫發(fā)現(xiàn)下調(diào)最顯著的miR-329-3p與PKA激酶的調(diào)節(jié)亞基prkarla,prkacb和催化亞基存在結(jié)合位點。與轉(zhuǎn)染陰性對照組質(zhì)粒相比,轉(zhuǎn)染了miR-329-3p質(zhì)粒后能顯著降低裝載野生型Prkarla、Prkacb序列的熒光素酶活性,而對陰性對照熒光素酶質(zhì)粒和裝載突變型Prkarla、Prkacb序列的熒光素酶活性沒有影響。4.使用Promega試劑盒檢測MC-LR染毒后GT1-7細胞中PKA的酶活改變,MC-LR染毒GT1-7細胞后激活PKA酶。四、結(jié)論1.MC-LR染毒GT1-7細胞后,使其miRNA表達譜發(fā)生改變,42種miRNA顯著上調(diào),59種miRNA顯著下調(diào)。其中,上調(diào)最明顯的是miR-544-3p,上調(diào)了 10.67倍。下調(diào)最明顯的是miR-329-3p,下調(diào)了 9.5倍。2.通過熒光素酶報告基因?qū)嶒炞C實PKA酶的調(diào)節(jié)亞基Prkarla和催化亞基Prkacb是miR-329-3p的靶基因。3.MC-LR染毒GT1-7細胞激活PKA酶。第二部分探究MC-LR激活PKA通路對GT1-7細胞合成GnRH的影響及分子機制一、目的探究MC-LR染毒GT1-7細胞激活PKA酶后,對GT1-7細胞合成GnRH的影響及其分子機制。二、方法1.不同濃度MC-LR不同時間點染毒GT1-7細胞,qPCR和Elisa分別檢測GnRH在轉(zhuǎn)錄水平和釋放水平的表達量變化。2.500nMMC-LR染毒GT1-7細胞0、0.25、0.5、1、3、6h,和0、10nM、100nM、500 nM MC-LR染毒GT1-7細胞48 h后,qPCR檢測Prkarla、Prkacb、c-Jun、c-Fos基因表達變化;Elisa檢測cAMP含量變化;Western Blot檢測PRKAR1A、PRKACB、c-Jun、c-Fos、CREB、p-CREB蛋白的表達量變化。3.PKA抑制劑H-89 2HCl(10μM)處理GT1-7細胞1h,500nMMC-LR染毒24h后,qPCR和Elisa分別檢測GnRH表達量變化,Westernblot檢測CREB、p-CREB蛋白的表達量變化。4.500nMMC-LR染毒GT1-7細胞6 h,染色質(zhì)免疫沉淀技術(shù)(CHIP)檢測p-CREB與c-Fos、c-Jun啟動子的結(jié)合含量變化,c-Fos、c-Jun與GnRH啟動子和增強子的結(jié)合含量變化。三、結(jié)果1.qPCR檢測MC-LR染毒GT1-7細胞后GnRH在轉(zhuǎn)錄水平的表達量,結(jié)果表明:隨著MC-LR染毒濃度和染毒時間的增加,GnRH的合成受到抑制。Elisa檢測GnRH在釋放水平表達量發(fā)現(xiàn):低濃度(5 nM)MC-LR刺激GT1-7細胞釋放GnRH,高濃度(500 nM)抑制GnRH的釋放。2.qPCR 檢測發(fā)現(xiàn):MC-LR 染毒 GT1-7 細胞后,Prkarla、Prkacb、c-Jun、c-Fos 基因表達上調(diào),提示PKA通路激活。Westernblot檢測發(fā)現(xiàn):MC-LR染毒GT1-7細胞后,PRKAR1A、PRKACB、c-Jun、c-Fos、p-CREB 蛋白表達量上調(diào),進一步驗證PKA通路激活。3.PKA抑制劑處理GT1-7細胞1 h后,500 nM MC-LR染毒24 h發(fā)現(xiàn):加入抑制劑后,MC-LR對GnRH的抑制作用消除。4.CHIP實驗發(fā)現(xiàn):MC-LR染毒GT1-7細胞后,p-CREB與c-Fos、c-Jun啟動子的結(jié)合量增多,c-Fos、c-Jun與GnRH啟動子和增強子的結(jié)合量增多。四、結(jié)論1.MC-LR染毒GT1-7細胞,導致細胞活性下降,抑制GnRH的合成,低濃度(5nM)MC-LR促進GnRH的釋放,高濃度(500 nM)抑制GnRH的釋放。2.MC-LR通過激活PKA-p-CREB-c-Fos/c-Jun這條通路抑制GnRH的合成。第三部分調(diào)控miR-329-3p對MC-LR影響GnRH合成的干預作用一、目的調(diào)控miR-329-3p的表達,探討miR-329-3p對MC-LR影響GnRH合成的干預作用。二、方法1.采用脂質(zhì)體轉(zhuǎn)染外源性 miR-329-3p mimics、inhibitors 或 miRNA negative control進入GT1-7細胞,qPCR檢測miR-329-3p和GnRH mRNA表達水,采用Western檢測 PRKACA、PRKACB、p-CREB、CREB、c-Fos、c-Jun 的含量變化。2.GT1-7 細胞轉(zhuǎn)染 miR-329-3p mimic,同時染毒 500 nM MC-LR,采用 qPCR 法檢測GnRH mRNA表達水平;采用Western blot實驗技術(shù)檢測PRKACA、PRKACB、p-CREB、CREB、c-Fos、c-Jun 蛋白表達水平。三、結(jié)果1.與對照組相比,miR-329-3p mimics后能顯著升高miR-329-3p的表達量,PRKACA、PRKACB表達量隨之下降,促進GnRH的轉(zhuǎn)錄;而miR-329-3p inhibitor組miR-329-3p表達量顯著下調(diào),PRKACA、PRKACB表達量顯著上調(diào),抑制GnRH的轉(zhuǎn)錄。2.與對照組相比,同時轉(zhuǎn)染miR-329-3pmimics和MC-LR組可以消除MC-LR引起的 PRKACA、PRKACB、c-Jun、c-Fos、p-CREB 的上調(diào),有效逆轉(zhuǎn) MC-LR 對GnRH合成的抑制作用。四、結(jié)論1.miR-329-3p可以調(diào)控PKA酶的調(diào)節(jié)亞基Prkarla和催化亞基Prkacb的表達。2.調(diào)控miR-329-3p的表達可以影響GnRH合成,上調(diào)miR-329-3p的表達可以促進GnRH的合成,下調(diào)miR-329-3p的表達抑制GnRH的合成。3.過表達miR-329-3p抑制了 PKA通路的激活,有效逆轉(zhuǎn)了因MC-LR激活PKA通路引起的對GnRH合成的抑制作用。
[Abstract]:Microcystins (MCs) is a kind of natural toxin produced by fresh water cyanobacteria, which is composed of seven amino acids. It has strong liver toxicity, neurotoxicity, nephrotoxicity and gastrointestinal toxicity. The group of [1-3]. is the first to find that MCs has male reproductive toxicity. In our previous study, we have proved MC-LR by acute and chronic experiments. One of the most widely distributed and most toxic MCs isomers in the water body caused the level of testosterone in the body of the male rats. The decrease of the testosterone level was 60% ~ 80%[4]. in the male animals, and the Leydig cells in the testis were an important place for the synthesis of testosterone. In vitro and in vitro experiments showed that MC-LR could not enter interstitial cells and had no interstitial cells. The synthetic testosterone of [5]. interstitial cells was regulated by the hypothalamus adenohypophysis axis. The GnRH secreted by GnRH neurons in the hypothalamus indirectly regulates the synthesis of testosterone in the stromal cells. The previous results showed that MC-LR could effectively enter the hypothalamus, the amount of [6]. in the GnRH was known, and the GnRH was secreted by the GnRH neurons in the hypothalamus. Due to GnRH, The number of neurons is small and scattered in the hypothalamus. We use GT1-7 cells for subsequent mechanisms to study the stable secretion of GnRH by.GT1-7 cells. It is the ideal cell line of GnRH neurons to study the molecular mechanism of the inhibition of GnRH synthesis after MC-LR enters GT1-7 cells. The full text is divided into three parts: the first part MC-LR to GT1-7 fines The effect of miRNA expression profile 1, aim to explore the effect of MC-LR on the miRNA expression profiles in GT1-7 cells and to screen the miRNA. two related to GnRH synthesis. Methods 1.GT1-7 cells were divided into 10 groups to detect all indexes, namely Control, 1nM, 10nM, 100nM, 500nM, 5 micron, 10 micron, 50 micron, 1000 micron, 1000 micron, and 1000 micron. The effects of MC-LR on the morphology, vitality and survival rate of GC-1 cells were detected, and the optimal concentration and time of GT1-7 cells were confirmed. MiRNA hybridization chip was used to select the best dyeing conditions for miRNA chip selection. After screening the MC-LR infected GT1-7 cells, the miRNA. expression in the cells was tested by qPCR experimental technique to verify the reliability of the chip. 4.miR-329-3p target gene prediction and verification: using the bioinformatics software such as miRanda, Targetscan and microRNA.org to predict the target genes of differential miRNA, and to screen out miRNA., which is related to GnRH synthesis, to construct the luciferase reporter gene recombinant plasmid containing Prkarla, Prkacb and miR-329-3p complementary pairs of base sequence, using lipid. Plasmid transfected exogenous miR-329-3p or negative control and recombinant plasmid or empty plasmid into 293T cells to verify the relationship between miR-329-3p and Prkarla, Prkacb,.5. using Promega kit to detect the activity of PKA enzyme activity in GT1-7 cells after MC-LR infected by MC-LR. Three. Results 1. With the increase of R concentration and time, the activity of cells decreased in.100nM-100 mu M group, and the activity of GT1-7 cells decreased significantly. The morphology of cells in the 0-1000 nM group was not obviously changed. The cells in the group of 10 and 100 mu M were round and suspended in the medium of.2.MC-LR to miRNA expression of GT1-7 cells in the medium. Effect: 500 nM MC-LR infected GT1-7 cells 48 h. By comparing the changes of miRNA in GT1-7 cells before and after exposure to GT1-7 cells, 101 kinds of miRNA were found to be significantly altered, of which 42 were significantly up-regulated and 59 were significantly down. The most obvious up regulation was miR-544-3p, up to 10.67 times. The most obvious downregulation was 9.5 times the expression difference of 9.5 times.QPCR. The miRNAs results are consistent with the chip.3.. The binding sites of the most significant miR-329-3p and PKA kinase's regulatory subunits prkarla, prkacb and the catalytic subunits are down regulated by a comprehensive comparison of different database findings. The transfection of miR-329-3p plasmid to the plasmid transfected with a negative control group can significantly reduce the fluorescence loading of the wild type Prkarla and the Prkacb sequence. The activity of the protease, while the luciferase activity of the negative control luciferase plasmid and the loading mutant Prkarla, the Prkacb sequence did not affect the.4. enzyme activity change in the GT1-7 cell of GT1-7 cells infected with MC-LR by the Promega kit, and MC-LR was infected with the GT1-7 cell to activate the PKA enzyme. Four. The 42 kinds of miRNA were significantly up-regulated and 59 kinds of miRNA were down significantly down. Among them, the most obvious up-regulation was miR-544-3p, up 10.67 times. The most obvious downregulation was miR-329-3p, and 9.5 times down regulated by fluorescein reporter gene experiment confirmed that the PKA subunit Prkarla and the catalytic subunit Prkacb are miR-329-3p target genes.3.MC-LR. 7 cells activated the PKA enzyme. Second to explore the effect of MC-LR activation of PKA pathway on the synthesis of GnRH in GT1-7 cells and the molecular mechanism. The purpose of this study is to explore the effect of MC-LR on the activation of PKA enzyme in GT1-7 cells and the molecular mechanism of GT1-7 cells to synthesize GnRH. Two NRH at the transcriptional level and the expression level of the release level.2.500nMMC-LR 0,0.25,0.5,1,3,6h, and 0,10nM, 100nM, 500 nM MC-LR infected GT1-7 cell 48 h. The expression of.3.PKA inhibitor H-89 2HCl (10 mu M) was used to treat GT1-7 cell 1H. After 500nMMC-LR was infected with 24h, qPCR and Elisa detected the GnRH expression change. Content changes, c-Fos, c-Jun and GnRH promoter and enhancer binding content changes. Three, 1.qPCR detection of MC-LR infected GT1-7 cells after GnRH at the transcriptional level of expression, the results showed that as MC-LR dye concentration and time increased, GnRH synthesis was inhibited.Elisa GnRH in the release level of expression of the discovery: low concentration (5 N) MC-LR stimulates GT1-7 cells to release GnRH, and high concentration (500 nM) inhibits the release of GnRH by.2.qPCR detection. One step verified that after the PKA pathway activated the.3.PKA inhibitor to treat GT1-7 cells 1 h, the 500 nM MC-LR poisoning 24 h found that the inhibition of MC-LR to GnRH was eliminated after the addition of the inhibitor, and the.4.CHIP experiment found that the binding amount of the promoter was increased after the MC-LR poisoned GT1-7 cells. Four, conclusion 1.MC-LR infected GT1-7 cells, causing cell activity to decrease, inhibit the synthesis of GnRH, low concentration (5nM) MC-LR promotes GnRH release, high concentration (500 nM) inhibits GnRH release.2.MC-LR by activating PKA-p-CREB-c-Fos/c-Jun this pathway inhibits GnRH synthesis. The third part regulates the interference effect of miR-329-3p on the synthesis. Objective to regulate the expression of miR-329-3p and to explore the interference effect of miR-329-3p on the synthesis of GnRH by MC-LR. Two, method 1. transfection of exogenous miR-329-3p mimics with liposomes, inhibitors or miRNA negative control into GT1-7 cells. .2.GT1-7 cells were transfected to miR-329-3p mimic and 500 nM MC-LR, qPCR method was used to detect the mRNA expression level of GnRH, and Western blot experimental technique was used to detect the expression of PRKACA. The expression of PRKACA and PRKACB decreased and promoted the transcription of GnRH, while the expression of miR-329-3p in the miR-329-3p inhibitor group was significantly down, the expression of PRKACA, PRKACB was significantly up-regulated, and the transcriptional.2. of the GnRH was compared with that of the control group. Up regulation of B can effectively reverse the inhibitory effect of MC-LR on GnRH synthesis. Four, conclusion 1.miR-329-3p can regulate the expression of PKA regulating subunit Prkarla and catalytic subunit Prkacb to regulate the expression of miR-329-3p, which can affect the synthesis of GnRH. Up regulation of miR-329-3p expression can promote the synthesis of GnRH. 3. overexpression of miR-329-3p inhibits activation of PKA pathway and reverses the inhibition of GnRH synthesis induced by MC-LR activation of PKA pathway.

【學位授予單位】：南京大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R99
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本文編號：1874011