【作者】 王静；

【导师】 韩晓冬；

【作者基本信息】 南京大学 ， 药物化学， 2017， 硕士

【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。更多还原

【Abstract】 Microcystins(MCs),which have strong toxicity on liver,nerve,kidney and gastrointestinal,are natural toxins composed of seven amino acids and secreted by freshwater cyanobacteria in the process of blooming.In previous studies of our laboratory,it was discovered that MC-LR has strong male reproductive toxicity for the first time.Previously,we have found that MC-LR(the most abundant and the most toxic isoform of microcystins)can cause the significant reduction of serum testosterone by 60%to 80%according to both acute and chronic experiments.Leydig cells are the main place for testosterone biosynthesis in male.MC-LR cannot enter into the leydig cells without damaging to them by both in vitro and in vivo experiments.The testosterone synthesis in leydig cells are affected by the hypothalamic-pituitary-gonad axis.The GnRH neurons in hypothalamus regulate the synthesis of testosterone indirectly.Based on previous work,MC-LR can get into the hypothalamus and reduce male animals’testosterone synthesis and secretion.Due to the cellular complexity of the brain and the absence of cultured model systems representing differentiated central nervous system neurons,we use the GT1-7 cells for further molecular mechanisms study.GT1-7 cells are neuronal in physiology and morphology and secrete GnRH steadily in a pulsatile fashion,as it does in the intact hypothalamus.Organic anion transporting polypeptides(Oatps)constitute the specific transport protein that can mediate the transportation of MCs from the blood into the cell.Oatp1a5 is the pivotal protein that transport MC-LR into GT1-7 cells based on our previous study.Our research will clarify the mechanism about MC-LR-induced the suppression of Gonadotropin-releasing hormone synthesis in GT1-7 cells.The thesis is divided into three parts:Part I:The effects of MC-LR on miRNA profiling expression in GT1-7ObjectiveTo study the effects of MC-LR on miRNA profiling expression in GT1-7 cells and screen the miRNA associated with the synthesis of GnRH.Methods1.After exposure to MC-LR for different concentrations and time,CCK-8 method was used to determine the viability of GT1-7 cells.2.In order to choose the optimal concentration of MC-LR for miRNA array,cell viability and morphology were recorded in GT1-7 cells after exposure to a range of concentrations.We observed cell morphology under the microscope;We use CCK-8 method to examine the MC-LR toxicology on cell viability.3.We use the Hybrid chip microarray to detect the altertion miRNA of GT1-7 cells after exposure to 500 nM MC-LR for 48h.miRanda、Targetscan and microRNA.org were applied to predict the targets of the alteration miRNA in order to find the miRNA associated with the suppression of GnRH.qRT-PCR was used to validate the array data.4.The prediction and variation of the targets of miR-329-3p1)Using bioinformatics software,such as miRanda、Targetscan and microRNA.org,we can predict the target gene of the significantly changed miRNA and filter out the miRNAs associated with the synthesis of GnRH.Taqman probe is used to test miR-329-3p again.2)The 3’-UTRs of mouse Prkarla and Prkacb encompassing the conserved miR-329-3p response elements were cloned into the pMIR-REPORT vector.These luciferase reporters were transiently transfected,together with miR-329-3p-mimic,control-mimic,miR-329-3p-inhibitor,and control inhibitor into 293T cells which were a model system for the validation of miRNA target genes.5.The Promega kit is used to test the change of PKA enzyme activity in GT1-7 cells treated by MC-LR.Results1.In CCK-8 tests,Cell viability was decreased after exposure to MC-LR in does-dependent and time-dependent manner.Cell viability was significantly decreased after exposure to MC-LR for 48 h at a concentration of 500 nM or higher.2.GT1-7 cells were treated by 500 nM MC-LR for 48h,comparing with the control group,miRNA expression profiling revealed 101 miRNAs with substantial changes in GT1-7 cells.Of the 101 miRNAs with changes in their expression,42 were significantly up-regulated while 59 were evidently downregulated.The expression of all the selected miRNAs was consistent with the array data suggesting the reliability of the array.3.The prediction and variation of the targets of miR-329-3p.1)miR-329-3p was speculated to be the candidate miRNA that targets Prkar1a and Prkacb using computational prediction programs.2)Compared with transfection negative controls,the plasmid group and transfection miR-329-3p after plasmid can significantly reduce the loading of wild type Prkar1a and Prkacb sequence of fluorescein enzyme activity,but the negative control luciferin enzyme plasmid and load mutant Prkarla and Prkacb sequence of fluorescein had no effect on enzyme activity.4.Comparing with the control group,GTl-7 cells treated by MC-LR evoke the PKA enzyme.Conclusions1.The Prkar1a、Prkacb are the target genes of miR-329-3p confirmed by luciferase reporters.2.GT1-7 cells treated by MC-LR evoke the PKA enzyme.Part II:To study the effect and mechanism of the activation of PKA enzyme in GT1-7 cells by MC-LR.ObjectiveStudy the effect and mechanism of the activation of PKA enzyme in GT1-7 cells by MC-LR.Methods1.After exposure to MC-LR for different concentrations and time,GnRH mRNA level was determined using qPCR and GnRH concentration in supernatant was tested by ELISA.2.GT1-7 cells were exposed to 500 nM MC-LR for 0,0.25,0.5,1,3,6 h.Expression levels of Prkarla、Prkacb.c-Jun、c-Fos were determined by qPCR.cAMP and was tested by ELISA.The protein expression of Prkar1a、Prkacb、c-Jun、c-Fos、creb、p-creb were determined by Western blot.3.After exposure to the PKA inhibitor H-89 2HCl(10μM)for 1h individually,GT1-7 cells were dealt with 500 nM MC-LR for 24h,GnRH mRNA level was determined using qPCR.The protein expression of creb、p-creb were determined by Western blot.4.After exposure to 500 nM MC-LR for 6h,CHIP assays were performed to assess the interaction of p-CREB with c-Fos and c-Jun promoter,the interaction of c-Fos and c-Jun with GnRH promoter as well as enhancer.Results1.Q-PCR indicated GnRH transcription level decreased significantly as MC-LR concentration and exposure time increased.After exposure of MC-LR to GT1-7,we found as MC-LR concentration increased,GnRH level in the cell culture medium exhibited an increase-followed-by-decrease trend.2.After exposure of GT1-7 to MC-LR,the qPCR results showed that the expression of Prkar1a、Prkacb、c-Jun、c-Fos were upregulated indicating that PKA pathway was evoked;the Westernblot showed that the protein expression of Prkarl a、Prkacb、c-Jun、c-Fos、p-CREB were upregulated further confirming that PKA pathway was evoked.3.Pretreatment with PKA inhibitors abolished the suppression of GnRH induced by MC-LR.4.ChIP assay studies demonstrated that MC-LR increased p-CREB/c-Jun,p-CREB/c-Fos interaction,and c-Jun/GnRH promoter and enhancer interaction,and c-Fos/GnRH promoter and enhancer interaction.Conclusions1.MC-LR showed significant toxicity on GT1-7 cells.Cell viability was decreased and GnRH synthesis was suppressed after exposure to MC-LR.2.MC-LR evokes the PKA pathway in GT1-7 cells.Suppressing the pathway can abolished suppression of GnRH induced by MC-LR.Part III:The effect of regulating miR-329-3p on MC-LR-induced the suppression of GnRHObjectiveRegulate the expression of miR-329-3p to study the effects of MC-LR-induced the suppression of GnRH.Methods1.miR-329-3p affect the synthesis of GnRH by Prkarla and PrkacbTransfecting miR-329-3p-mimic,control-mimic,miR-329-3p-inhibitor,and control inhibitor into GT1-7 cells.miR-329-3p,Prkar1a、Prkacb and GnRH mRNA level was determined using qPCR.The protein expression of Prkar1a、Prkacb were determined by Western blot.2.Overexpression miR-329-3p can reverse the suppression of GnRH synthesis induced by MC-LRGT1-7 cells were transfected with miR-329-3p-mimic,control-mimic,miR-329-3p inhibitor or control-inhibitor followed by incubation for 24 h in the presence of MC-LR.miR-329-3p,Prkar1a、Prkacb and GnRH mRNA level was determined using qPCR.The protein expression of Prkar1a、Prkacb、c-Jun、c-Fos、creb、p-creb were determined by Western blot.Results1.miR-329-3p affect the synthesis of GnRH by Prkar1a and PrkacbCompared with the control group,transfection miR-329-3p mimics can significantly increase the amount of miR-329-3p,reduce the mRNA level of Prkar1、Prkacb and GnRH.Transfection miR-329-3p inhibitors causes the country results.2.Overexpression miR-329-3p can reverse the suppression of GnRH synthesis induced by MC-LRCompared with the control group,transfection miR-329-3p and MC-LR can abolish the increase of Prkar1a、Prkacb、c-Jun、c-Fos、p-CREB induced by MC-LR,and reverse thesuppression of GnRH caused by MC-LR.Conclusions1.miR-329-3p affect Prkar1a、Prkacb in transcription and translation level inhibits the expression of Prkarla、Prkacb.2.Overexpression of miR-329-3p can suppress the expression of Prkar1a、Prkacb,thus suppress the activation of PKA pathway,and abolish the suppression of GnRH caused by MC-LR.更多还原