微囊藻毒素（MC-LR）抑制GT1-7细胞合成GnRH的分子机制探究

发布时间：2018-05-11 12:53

  本文选题：MC-LR + GT1-7　； 参考：《南京大学》2017年硕士论文

【摘要】：微囊藻毒素(Microcystins,MCs)是淡水蓝藻产生的一类由七个氨基酸组成的天然毒素,具有强烈的肝毒性、神经毒性、肾毒性和胃肠道毒性[1-3]。本课题组率先发现MCs具有雄性生殖毒性。我们在前期研究中,分别通过急性和慢性实验证明了MC-LR(水体中分布最广、毒性最强的一种MCs异构体)染毒雄性大鼠后,引起机体睾酮水平明显下降,其下降幅度为60%～80%[4]。在雄性动物体内,睾丸间质细胞是合成睾酮的重要场所。而体内体外实验均表明,MC-LR不能进入间质细胞,对间质细胞没有明显的损伤作用[5]。间质细胞合成睾酮受下丘脑-腺垂体-睾丸轴的调控。下丘脑GnRH神经元分泌的GnRH间接调控间质细胞合成睾酮。前期结果表明,MC-LR能够有效进入下丘脑组织,下调GnRH的量[6]。已知,GnRH由下丘脑中GnRH神经元分泌。由于GnRH神经元数量少,在下丘脑分布零散,我们使用GT1-7细胞进行后续的机制研究。GT1-7细胞能稳定的分泌GnRH,是研究GnRH神经元的理想细胞株[7-8]。本研究探讨MC-LR进入GT1-7细胞后,抑制GnRH合成的分子机制。全文分为三部分:第一部分MC-LR对GT1-7细胞miRNA表达谱的影响一、目的探究MC-LR对GT1-7细胞miRNA表达谱的影响,筛选与GnRH合成相关的miRNA。二、方法1.GT1-7细胞均分10组进行各项指标的检测,即Control、1nM、10nM、100nM、500nM、1μM、5μM、10μM、50μM、1000μM MC-LR组。2.光镜下观察细胞形态,采用CCK-8法,检测MC-LR对GC-1细胞形态,活力和存活率的影响,确认GT1-7细胞最佳的染毒浓度和时间,为进行miRNA芯片筛选选择最佳染毒条件。3.采用miRNA杂交芯片法,筛选出MC-LR染毒GT1-7细胞后,细胞中表达变化的miRNA。采用qPCR实验技术验证芯片的可靠性。4.miR-329-3p靶基因的预测和验证:采用miRanda、Targetscan及microRNA.org等生物信息学软件预测差异变化miRNA的靶基因,借此筛选出与GnRH合成相关的miRNA。分别构建含Prkarla、Prkacb与miR-329-3p互补配对碱基序列的荧光素酶报告基因重组质粒,采用脂质体转染外源性miR-329-3p或negative control和重组质粒或空载质粒进入293T细胞,验证miR-329-3p与Prkarla、Prkacb之间的作用关系。5.使用Promega试剂盒检测MC-LR染毒后GT1-7细胞中PKA的酶活性是否改变。三、结果1.采用CCK-8法检测细胞活力发现:随着MC-LR染毒浓度和染毒时间的增加,细胞活性呈现下降趋势。100nM-100μM组染毒48h,GT1-7细胞活力显著降低。光镜下观察细胞的形态发现:0-1000 nM组细胞形态未明显改变,10 μM和100 μM组细胞呈圆形并悬浮于培养基中。2.MC-LR对GT1-7细胞miRNA表达谱的影响:500 nM MC-LR染毒GT1-7细胞48 h,通过对比GT1-7细胞染毒前后miRNA的变化,发现101种miRNA发生明显改变,其中42种显著上调,59种显著下调。其中,上调最明显的是miR-544-3p,上调了 10.67倍。下调最明显的是miR-329-3p,下调了 9.5倍。qPCR证实表达差异的miRNAs结果与芯片一致。3.通过综合比对不同数据库发现下调最显著的miR-329-3p与PKA激酶的调节亚基prkarla,prkacb和催化亚基存在结合位点。与转染阴性对照组质粒相比,转染了miR-329-3p质粒后能显著降低装载野生型Prkarla、Prkacb序列的荧光素酶活性,而对阴性对照荧光素酶质粒和装载突变型Prkarla、Prkacb序列的荧光素酶活性没有影响。4.使用Promega试剂盒检测MC-LR染毒后GT1-7细胞中PKA的酶活改变,MC-LR染毒GT1-7细胞后激活PKA酶。四、结论1.MC-LR染毒GT1-7细胞后,使其miRNA表达谱发生改变,42种miRNA显著上调,59种miRNA显著下调。其中,上调最明显的是miR-544-3p,上调了 10.67倍。下调最明显的是miR-329-3p,下调了 9.5倍。2.通过荧光素酶报告基因实验证实PKA酶的调节亚基Prkarla和催化亚基Prkacb是miR-329-3p的靶基因。3.MC-LR染毒GT1-7细胞激活PKA酶。第二部分探究MC-LR激活PKA通路对GT1-7细胞合成GnRH的影响及分子机制一、目的探究MC-LR染毒GT1-7细胞激活PKA酶后,对GT1-7细胞合成GnRH的影响及其分子机制。二、方法1.不同浓度MC-LR不同时间点染毒GT1-7细胞,qPCR和Elisa分别检测GnRH在转录水平和释放水平的表达量变化。2.500nMMC-LR染毒GT1-7细胞0、0.25、0.5、1、3、6h,和0、10nM、100nM、500 nM MC-LR染毒GT1-7细胞48 h后,qPCR检测Prkarla、Prkacb、c-Jun、c-Fos基因表达变化;Elisa检测cAMP含量变化;Western Blot检测PRKAR1A、PRKACB、c-Jun、c-Fos、CREB、p-CREB蛋白的表达量变化。3.PKA抑制剂H-89 2HCl(10μM)处理GT1-7细胞1h,500nMMC-LR染毒24h后,qPCR和Elisa分别检测GnRH表达量变化,Westernblot检测CREB、p-CREB蛋白的表达量变化。4.500nMMC-LR染毒GT1-7细胞6 h,染色质免疫沉淀技术(CHIP)检测p-CREB与c-Fos、c-Jun启动子的结合含量变化,c-Fos、c-Jun与GnRH启动子和增强子的结合含量变化。三、结果1.qPCR检测MC-LR染毒GT1-7细胞后GnRH在转录水平的表达量,结果表明:随着MC-LR染毒浓度和染毒时间的增加,GnRH的合成受到抑制。Elisa检测GnRH在释放水平表达量发现:低浓度(5 nM)MC-LR刺激GT1-7细胞释放GnRH,高浓度(500 nM)抑制GnRH的释放。2.qPCR 检测发现:MC-LR 染毒 GT1-7 细胞后,Prkarla、Prkacb、c-Jun、c-Fos 基因表达上调,提示PKA通路激活。Westernblot检测发现:MC-LR染毒GT1-7细胞后,PRKAR1A、PRKACB、c-Jun、c-Fos、p-CREB 蛋白表达量上调,进一步验证PKA通路激活。3.PKA抑制剂处理GT1-7细胞1 h后,500 nM MC-LR染毒24 h发现:加入抑制剂后,MC-LR对GnRH的抑制作用消除。4.CHIP实验发现:MC-LR染毒GT1-7细胞后,p-CREB与c-Fos、c-Jun启动子的结合量增多,c-Fos、c-Jun与GnRH启动子和增强子的结合量增多。四、结论1.MC-LR染毒GT1-7细胞,导致细胞活性下降,抑制GnRH的合成,低浓度(5nM)MC-LR促进GnRH的释放,高浓度(500 nM)抑制GnRH的释放。2.MC-LR通过激活PKA-p-CREB-c-Fos/c-Jun这条通路抑制GnRH的合成。第三部分调控miR-329-3p对MC-LR影响GnRH合成的干预作用一、目的调控miR-329-3p的表达,探讨miR-329-3p对MC-LR影响GnRH合成的干预作用。二、方法1.采用脂质体转染外源性 miR-329-3p mimics、inhibitors 或 miRNA negative control进入GT1-7细胞,qPCR检测miR-329-3p和GnRH mRNA表达水,采用Western检测 PRKACA、PRKACB、p-CREB、CREB、c-Fos、c-Jun 的含量变化。2.GT1-7 细胞转染 miR-329-3p mimic,同时染毒 500 nM MC-LR,采用 qPCR 法检测GnRH mRNA表达水平;采用Western blot实验技术检测PRKACA、PRKACB、p-CREB、CREB、c-Fos、c-Jun 蛋白表达水平。三、结果1.与对照组相比,miR-329-3p mimics后能显著升高miR-329-3p的表达量,PRKACA、PRKACB表达量随之下降,促进GnRH的转录;而miR-329-3p inhibitor组miR-329-3p表达量显著下调,PRKACA、PRKACB表达量显著上调,抑制GnRH的转录。2.与对照组相比,同时转染miR-329-3pmimics和MC-LR组可以消除MC-LR引起的 PRKACA、PRKACB、c-Jun、c-Fos、p-CREB 的上调,有效逆转 MC-LR 对GnRH合成的抑制作用。四、结论1.miR-329-3p可以调控PKA酶的调节亚基Prkarla和催化亚基Prkacb的表达。2.调控miR-329-3p的表达可以影响GnRH合成,上调miR-329-3p的表达可以促进GnRH的合成,下调miR-329-3p的表达抑制GnRH的合成。3.过表达miR-329-3p抑制了 PKA通路的激活,有效逆转了因MC-LR激活PKA通路引起的对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.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R99
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本文编号：1874011