Egr-1 siRNA抑制吗啡戒断后PC12细胞Egr-1基因表达及细胞凋亡
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摘要
研究背景
长期使用阿片类药物可以影响大脑多方面的功能并引起耐受、依赖和成瘾,最终导致身心健康损害和社会活动障碍,对人体和社会造成不可估量的损失。由于绝大多数成瘾患者仍得不到充分有效的治疗,因此探索阿片类药物成瘾的中枢机制,将有助于提高戒毒治疗效果,最终根除成瘾并能预防成瘾的复发。但令人遗憾的是,药物依赖和成瘾的机制仍不清楚。
吗啡慢性处理和吗啡戒断可以引起神经细胞的结构损伤以及诱导神经细胞凋亡,神经细胞凋亡可引起的脑内神经细胞基因异常表达、神经细胞的功能减退以及神经可塑性变化。而目前研究表明,脑内神经细胞基因异常表达,神经细胞功能减退,以及神经可塑性变化可能是阿片类药成瘾发生的主要原因之一。因此,慢性吗啡处理或吗啡戒断后细胞凋亡可能参与了阿片类药物依赖和成瘾。
Egr-1基因又称为zif268、NGFI-A和Krox-24,属于即刻-早期基因类,编码转录因子蛋白EGR-1,在大脑神经元内有着广泛的表达。EGR-1蛋白作为转录因子,调控着诸多靶基因的表达,并且作为神经细胞内可诱导的转录因子,EGR-1蛋白通过调控其它基因,从而参与中枢神经系统的可塑性变化。有研究表明,Egr-1基因参与细胞的凋亡和几种药物的成瘾,但尚不清楚其是否也参与了阿片类药物成瘾以及具体机制。
本实验首先观察不同浓度谷氨酸、吗啡、咪达唑仑、氯胺酮和纳络酮等药物对Egr-1基因表达的影响,探讨Egr-1基因表达的药理学机制。接着设计3条针对Egr-1 mRNA的siRNA,并甲基化siRNA加强其稳定性,然后通过转染PC12细胞和C6细胞,筛选出有效的siRNA。最后,把判定有效的甲基化siRNA转染到吗啡慢性处理的PC12细胞,观察其对吗啡急性戒断后细胞凋亡有无抑制效果。
本论文的内容分3个部分。
第一部分不同药物对Egr-1基因表达的影响目的探讨引起Egr-1基因表达的机制
方法采用单层细胞培养法,将大鼠PC12细胞或C6脑胶质瘤细胞株置于含10%胎牛血清和5%的马血清的DMEM-F12培养液中(含50U/ml青霉素和50 ug/ml链霉素),在5 %CO2孵箱37℃静置培养。观察0～10000μmol/L谷氨酸,以及0～1000μmol/L吗啡、咪达唑仑和纳络酮对PC12细胞或C6细胞Egr-1基因表达的影响,并判断诱导Egr-1基因表达的最佳谷氨酸浓度。另外,还观察吗啡、丙泊酚、氯胺酮和咪达唑仑对谷氨酸引起的Egr-1基因表达的影响。采用免疫组织化学方法在倒置显微镜和荧光显微镜观察EGR-1蛋白染色,Western-Blot显示EGR-1蛋白表达丰度。
结果在1～10000μmol/L的谷氨酸过刺激下,均可见EGR-1蛋白染色阳性,并且随着谷氨酸的浓度增加,免疫荧光细胞密度增加,荧光也越强,但达到1mmol/L以后,荧光细胞密度明显减少,荧光强度也有所下降。0～100μmol/L浓度吗啡作用下未见明显EGR-1蛋白染色阳性的细胞,而1000μmol/L时有少量EGR-1蛋白染色阳性的细胞。不同浓度的纳络酮作用下并未见EGR-1蛋白染色阳性的细胞。咪达唑仑100μmol/L以下时,未见明显EGR-1蛋白阳性染色,而达到100μmol/L时,出现了EGR-1蛋白染色阳性细胞。咪达唑仑、丙泊酚、氯胺酮和吗啡处理后,100μmol/L谷氨酸刺激下后的PC12细胞EGR-1蛋白染色阳性的细胞密度和荧光强度不同程度的下降; Western-Blot显示EGR-1蛋白表达丰度分别下降约70.2%,51.8%,84.6%和31.1%。结论低浓度急性吗啡和咪达唑仑处理并不诱导Egr-1基因的表达,而高浓度可引起Egr-1基因表达。谷氨酸能诱导Egr-1基因表达,其中以浓度100μmol/L最为明显。氯胺酮、吗啡、咪达唑仑和丙泊酚均能不同程度抑制谷氨酸引起的Egr-1基因表达。
第二部分Egr-1 siRNA对Egr-1基因表达的抑制
目的设计并筛选出高效能的抑制Egr-1基因表达的siRNA。
方法设计并合成3条甲基化Egr-1siRNA, siRNA-Ⅰ:正义链(5'-3') CCAACAGUGGCAACACUUU dTdT,反义链(3'-5')dTdT GGUUGUCACCGUUGUGAAA;siRNA-Ⅱ:正义链(5'-3') GACUUAAAGGCUCUUAAUA dTdT,反义链(3'-5'):dTdT -CUGAAUUUCCGAGAAUUAU;siRNA-Ⅲ:正义链(5'-3') GGACAAGAAAGCAGACAAA dTdT,反义链(3'-5')dTdT CCUGUUCUUUCGUCUGUUU。转染不同浓度的FAM-siRNA到PC12细胞,在倒置显微镜和荧光显微镜下观察转染效果。转染不同浓度EGFP–siRNA到含有pEGFP的PC12细胞,观察荧光抑制的情况。根据前面实验结果,50nM的siRNA-Ⅰ、Ⅱ、Ⅲ转染到PC12和C6细胞,在谷氨酸刺激下,通过Western-blot分析和RT-PCR判断Egr-1基因表达变化。
结果FAM-siRNA达50nM后,转染率已达80%～90%,随着浓度进一步增大,转染率和荧光强度并没有进一步提高。50nmol/L EGFP-siRNA抑制转染pEGFP-C1PC12细胞EGFP表达最为明显。Western-Blot显示50nM甲基化siRNA-Ⅰ、Ⅱ、Ⅲ可使PC12细胞EGR-1蛋白表达分别下降约85%、30%和78%;C6细胞EGR-1蛋白表达分别下降约90%、47%和85%。RT-PCR结果显示siRNA-Ⅰ、Ⅱ、Ⅲ分别下降PC12细胞Egr-1 mRNA数量的89%、47%和86%,C6细胞的92%、50%和85%。结论siRNA-Ⅰ和siRNA-Ⅲ可以充分下调细胞内Egr-1的表达,为实验需要的甲基化siRNA;FAM-siRNA和EGFP-siRNA可在检测转染效率的同时优化实验条件。
第三部分Egr-1 siRNA对吗啡戒断引起细胞凋亡的抑制作用及其机制研究
目的观察Egr-1 siRNA抑制吗啡戒断引起细胞凋亡的作用,探讨吗啡成瘾机制及治疗方法。
方法PC12细胞中加入100μmol/L吗啡, 48小时后给予10μmol/L纳络酮拮抗,利用倒置显微镜观测各组细胞形态及数量,然后EGR-1蛋白免疫荧光染色。接着转染50nM siRNAⅠ和siRNA-Ⅲ到PC12细胞,并设置N-siRNA-Ⅰ和N-siRNA-Ⅲ为对照以及阴性对照和阳性对照。同时慢性吗啡(100μmol/L)处理PC12细胞48小时,加入10μmol/L纳络酮,用MTT比色法观察PC12细胞的损害,应用FITC-AnnexinⅤ/PI流式凋亡检测试剂盒检测细胞凋亡率。
结果慢性吗啡处理组PC12细胞未见明显的荧光染色。吗啡慢性处理并急性戒断后1小时,即出现EGR-1蛋白阳性染色,但8小时后荧光染色细胞密度开始减少;24小时后光学显微镜观察细胞折光性能力减弱,细胞中颗粒增多,细胞皱缩,核固缩并断裂成数个,以及染色质凝集,大小不等的圆形颗粒及凋亡小体的形成。MTT法观察siRNA-Ⅰ和siRNA-Ⅲ能明显增加吗啡慢性处理急性戒断后细胞的存活率,差异有显著性意义(p<0.05);同时还观察到慢性吗啡处理组与空白对照组比较,生存率明显下降,差异有显著性意义(p<0.05);吗啡急性戒断24小时后,siRNA-Ⅰ和siRNA-Ⅲ分别能抑制27.7%和30.9%的细胞损害。与N-siRNA对照相比,流式细胞检测结果发现转染siRNA-Ⅰ和siRNA-Ⅲ的PC12细胞在吗啡急性戒断后,细胞凋亡水平分别下降了26.5%和20.3%。
结论Egr-1基因参与吗啡慢性处理和急性戒断后的细胞凋亡;下调Egr-1基因的表达,可降低吗啡戒断后细胞凋亡的发生率。
全文总结
1.EAAs、NMDA受体、GABAA受体、吗啡受体均参与调节Egr-1基因表达。
2.Egr-1基因参与吗啡慢性处理和急性戒断后的细胞凋亡。
3.下调Egr-1基因表达,可降低吗啡戒断后细胞凋亡的发生率。
4.Egr-1基因表达参与了阿片类药物的依赖和成瘾,具体机制可能与Egr-1基因表达增加诱发细胞凋亡有关。
Background
Chronic opiate applications lead to long-term impacts on many functions of the brain and induce tolerance, dependence and addiction. Addiction continues to exact enormous human and financial costs on society, but available treatments remain inadequate for most people. By analogy with other medical disorders, an improved understanding of the biological basis of addiction will lead to more effective treatments and eventually to cures and preventive measures. But it is regrettable that mechanisms of dependence and addiction are still not understood.
Previous research suggested that the abnormal gene expression in brain, impairment of neuronal cells and changes in the plasticity of neuron may play important roles in the development of opiate addiction. And chronic morphine treatment and morphine withdrawal cause apoptosis and injury of neuronal cells and other cells. So the neuronal injuries and impairments and changes in neuroplasticity in certain brain regions following chronic opiate treatment and opiate withdrawal could caused by apoptosis and have been implicated in the development of opioid dependence and addiction.
Egr-1, also called zif268, nerve growth factor-induced gene A (NGFI-A), belongs to the category of immediate early genes (IEG). It codes for a transcription factor protein, named EGR-1. Acting as a transcription factor, EGR-1 directly controls expression of other genes, which makes this protein an important object of studies aimed at understanding the orchestration of neuronal responses to a variety of stimuli. Specifically expressed in brain, Egr-1 can mediate cellular apoptosis and is involved in several drugs addiction. But it is unclear that Egr-1 is implicated in apoptosis resulted from chronic opiate treatment and opiate withdrawal, and the development of opioid addiction. The present study investigated the effect of acute morphine, glutamic acid, and midazolam administration, and chronic opiate treatment and opiate withdrawal on expression of Egr-1 in PC12 cells and C6 cells. Moreover, we design three Egr-1 siRNAs, and determine their functionality in PC12 cells and C6 cells. At last, selected potent Egr-1 siRNAs were transfected into PC12 cells. We study whether Egr-1siRNAs can inhibit cellular apoptosis and injuries following chronic opiate treatment and opiate withdrawal.
In this paper, the study was separated into three parts.
Part one The effect of different drugs on the expression of Egr-1. Aim To explore the effects of different drugs on levels of Egr-1 gene expression in PC12 cells or C6 cells.
Methods PC12 cells and Rat C6 glioma cells were seeded onto a 60-mm culture dish at a density of 1.5–2×106 cells/dish, and maintained in 5 ml of DMEM supplemented with 10% heat-inactivated fetal calf serum and 5% horse serum, 50 units/ml of penicillin, 50μg/ml of streptomycin at 37°C for 48 h in a humidified incubator containing 95% air–5% CO2 atmosphere. In the experiments testing whether glutamine acid, morphine, naloxone, and midazolam can induce EGR-1 protein expression, we treated the cells with 0-10000μM of glutamine acid, and 0-1000μM of morphine, naloxone, and midazolam. In the experiments testing the effects of pretreatment with morphine, naloxone, and midazolam were also present during treatment of the cells with glutamine acid. Immunocytochemical stain and western blot was used to detect the expression of Egr-1 gene.
Results EGR-1 protein staining were observed at 1-10000μM glutamine acid, and the optimal inducing concentration was 100μM. Furthermore, midazalam and morphine can induce expression of Egr-1 gene at a concentration more than 100μM and 1000μM respectively. Pretreatment of PC12 cells with 10μM midazolam, 100μM propofol, 100μM ketamine and 100μM morphine, the expression level of EGR-1 protein induced by glutamine acid significantly declined.
Conclusion Glutamine acid can induce expression of Egr-1 gene, but the level of Egr-1 gene expression induced by midazalam and morphine is determined by the concentrations of them. Moreover, midazolam, propofol, ketamine and morphine also could inhibit glutamine acid induced -EGR-1 protein expression.
Part two Inhibition of Egr-1 gene expression by Egr-1 siRNA. Aim To design Egr-1 siRNAs for selecting potent siRNAs and facilitating functional gene knockdown studies.
Methods Three Egr-1 siRNAs were designed and synthesized, siRNA-Ⅰ: sense, (5'-3') CCAACAGUGGCAACACUUUdTdT, antisense, (3'-5')dTdTGGUUGUCAC -CGU UG UGAAA; siRNA-Ⅱ:sense, (5'-3') GACUUAAAGGCUCUUAAUA dTdT; antisense, (3'-5')dTdT CUGAAUUUCCGAGAAUUAU; siRNA-Ⅲ: sense, (5'-3') GGAC AAGAAA GCAGACAAA dTdT, antisense, (3'-5')dTdTCCUGUUCUUUCGU–CUGUUU. In order to enhance serum and intracellular stability of siRNA, end modification of three terminal by chemical modifications were performed, such as 2’-OMe substitutions. Transfections of different concentrations of FAM-siRNA were performed with Lipofectamine TM 2000.
Threir transfection efficiency were determined by immunocytochemistry. In addition, 0-100μM EGFP–siRNAs were used to optimized transfection and test procedures in 6-well plates. Following transfection of three designed siRNA into PC12 cells and C6 cells at a concentration of 50nM, the level of glutamine acid induced-Egr-1 mRNA and EGR-1 protein were observed 48 later by RT-PCR and western-blot respectively.
Results Transfection efficiencies of FAM- siRNAs were 80%-90% at a concentration of 50nM, and increasing the concentration did not improve transfection effiencies. Two hours after transfection of EGFP–siRNAs into PC12 cells with pEGFP-C1, the suppressing expression of green fluorescence protein was observed. In addition, EGFP–siRNA had the most potent efficiency of inhibition at 50nM. 48 houes after siRNAⅠ,Ⅱ,Ⅲwere deliveried into PC12 cells and C6 cells, the levels of Egr-1 mRNA and EGR-1 protein induced by glutamine acid had reduced 89%, 47%, 86% and 85%, 30%, 78% in PC12 cells repectively. And in C6 cells, the reducing levels were 92%, 50%, 85% and 90%, 47%, 85% repectively.
Conclusion siRNAⅠ,Ⅲare able to down-regulate the expression of Egr-1 gene, and can be used to facilitate functional gene knockdown studies. In addition, EGFP-siRNA and FAM- siRNA can be used to determine the transfection efficiencies and optimized experimental procedures.
Part three The effect of Egr-1 siRNAs on cellular injuries and apoptosis following morphine withdrawal.
Aim To study the effect of Egr-1 siRNAs on cellular injuries and apoptosis following opiate withdrawal, and explore the mechanism of morphine addiction.
Methods After the cells were treated with 100μmol/ L morphine for 48 h, 10μmol/ L naloxone was used to produce acute withdrawal. The invert microscope was applied to observe morphology of the cells, and EGR-1 protein was detected by immunocytochemistry. Following transfection of siRNA, PC12 cells were treated with morphine for 48 h. After acute withdrawal with naloxone, thiazolyl blue tetrazolium bromide (MTT) assays were used to detect the proliferation of the cells and cells apoptosis were determined by AnnexinⅤ/PI flow cytometry.
Results Under invert microscope, the forms of cells were irregular and spindle, the chromatin concentrated into masses, the apoptosis bodies were formed 24h after morphine withdral. EGR-1 protein expression was appeared at 1h after withdrawal, reached peak at 8h and was detected even at 24h. MTT assays founded that mortality declined significantly (p<0.05 ), and apoptosis of PC12 cells who transfected siRNAⅠ,Ⅲreduced 26.5% and 20.3% respectively.
Conclusion Egr-1 gene is involved in apoptosis after chronic morphine treatment and acute morphine withidrawal. Down-regluation of Egr-1 gene can reduce apoptosis of cells induced by opiate withdrawal.
Summary
1. Glutamine acid could induce expression of Egr-1 gene, and midazalam, morphine, propofol and ketamine could inhibit its expression.
2. The designed siRNAⅠ,Ⅲcan down-regulate the expression of Egr-1 gene in PC12cells and C6 cells.
3. Egr-1 gene is involved in apoptosis after chronic morphine treatment and morphine withidrawal.
4. Down-regulation of Egr-1 gene can reduce apoptosis of cells induced by morphine withdrawal.
5. Apoptosis following chronic opiate treatment and opiate withdrawal could be caused by Egr-1 gene and could be implicated in the development of opioid dependence and addiction.
长期使用阿片类药物可以影响大脑多方面的功能并引起耐受、依赖和成瘾,最终导致身心健康损害和社会活动障碍,对人体和社会造成不可估量的损失。由于绝大多数成瘾患者仍得不到充分有效的治疗,因此探索阿片类药物成瘾的中枢机制,将有助于提高戒毒治疗效果,最终根除成瘾并能预防成瘾的复发。但令人遗憾的是,药物依赖和成瘾的机制仍不清楚。
吗啡慢性处理和吗啡戒断可以引起神经细胞的结构损伤以及诱导神经细胞凋亡,神经细胞凋亡可引起的脑内神经细胞基因异常表达、神经细胞的功能减退以及神经可塑性变化。而目前研究表明,脑内神经细胞基因异常表达,神经细胞功能减退,以及神经可塑性变化可能是阿片类药成瘾发生的主要原因之一。因此,慢性吗啡处理或吗啡戒断后细胞凋亡可能参与了阿片类药物依赖和成瘾。
Egr-1基因又称为zif268、NGFI-A和Krox-24,属于即刻-早期基因类,编码转录因子蛋白EGR-1,在大脑神经元内有着广泛的表达。EGR-1蛋白作为转录因子,调控着诸多靶基因的表达,并且作为神经细胞内可诱导的转录因子,EGR-1蛋白通过调控其它基因,从而参与中枢神经系统的可塑性变化。有研究表明,Egr-1基因参与细胞的凋亡和几种药物的成瘾,但尚不清楚其是否也参与了阿片类药物成瘾以及具体机制。
本实验首先观察不同浓度谷氨酸、吗啡、咪达唑仑、氯胺酮和纳络酮等药物对Egr-1基因表达的影响,探讨Egr-1基因表达的药理学机制。接着设计3条针对Egr-1 mRNA的siRNA,并甲基化siRNA加强其稳定性,然后通过转染PC12细胞和C6细胞,筛选出有效的siRNA。最后,把判定有效的甲基化siRNA转染到吗啡慢性处理的PC12细胞,观察其对吗啡急性戒断后细胞凋亡有无抑制效果。
本论文的内容分3个部分。
第一部分不同药物对Egr-1基因表达的影响目的探讨引起Egr-1基因表达的机制
方法采用单层细胞培养法,将大鼠PC12细胞或C6脑胶质瘤细胞株置于含10%胎牛血清和5%的马血清的DMEM-F12培养液中(含50U/ml青霉素和50 ug/ml链霉素),在5 %CO2孵箱37℃静置培养。观察0～10000μmol/L谷氨酸,以及0～1000μmol/L吗啡、咪达唑仑和纳络酮对PC12细胞或C6细胞Egr-1基因表达的影响,并判断诱导Egr-1基因表达的最佳谷氨酸浓度。另外,还观察吗啡、丙泊酚、氯胺酮和咪达唑仑对谷氨酸引起的Egr-1基因表达的影响。采用免疫组织化学方法在倒置显微镜和荧光显微镜观察EGR-1蛋白染色,Western-Blot显示EGR-1蛋白表达丰度。
结果在1～10000μmol/L的谷氨酸过刺激下,均可见EGR-1蛋白染色阳性,并且随着谷氨酸的浓度增加,免疫荧光细胞密度增加,荧光也越强,但达到1mmol/L以后,荧光细胞密度明显减少,荧光强度也有所下降。0～100μmol/L浓度吗啡作用下未见明显EGR-1蛋白染色阳性的细胞,而1000μmol/L时有少量EGR-1蛋白染色阳性的细胞。不同浓度的纳络酮作用下并未见EGR-1蛋白染色阳性的细胞。咪达唑仑100μmol/L以下时,未见明显EGR-1蛋白阳性染色,而达到100μmol/L时,出现了EGR-1蛋白染色阳性细胞。咪达唑仑、丙泊酚、氯胺酮和吗啡处理后,100μmol/L谷氨酸刺激下后的PC12细胞EGR-1蛋白染色阳性的细胞密度和荧光强度不同程度的下降; Western-Blot显示EGR-1蛋白表达丰度分别下降约70.2%,51.8%,84.6%和31.1%。结论低浓度急性吗啡和咪达唑仑处理并不诱导Egr-1基因的表达,而高浓度可引起Egr-1基因表达。谷氨酸能诱导Egr-1基因表达,其中以浓度100μmol/L最为明显。氯胺酮、吗啡、咪达唑仑和丙泊酚均能不同程度抑制谷氨酸引起的Egr-1基因表达。
第二部分Egr-1 siRNA对Egr-1基因表达的抑制
目的设计并筛选出高效能的抑制Egr-1基因表达的siRNA。
方法设计并合成3条甲基化Egr-1siRNA, siRNA-Ⅰ:正义链(5'-3') CCAACAGUGGCAACACUUU dTdT,反义链(3'-5')dTdT GGUUGUCACCGUUGUGAAA;siRNA-Ⅱ:正义链(5'-3') GACUUAAAGGCUCUUAAUA dTdT,反义链(3'-5'):dTdT -CUGAAUUUCCGAGAAUUAU;siRNA-Ⅲ:正义链(5'-3') GGACAAGAAAGCAGACAAA dTdT,反义链(3'-5')dTdT CCUGUUCUUUCGUCUGUUU。转染不同浓度的FAM-siRNA到PC12细胞,在倒置显微镜和荧光显微镜下观察转染效果。转染不同浓度EGFP–siRNA到含有pEGFP的PC12细胞,观察荧光抑制的情况。根据前面实验结果,50nM的siRNA-Ⅰ、Ⅱ、Ⅲ转染到PC12和C6细胞,在谷氨酸刺激下,通过Western-blot分析和RT-PCR判断Egr-1基因表达变化。
结果FAM-siRNA达50nM后,转染率已达80%～90%,随着浓度进一步增大,转染率和荧光强度并没有进一步提高。50nmol/L EGFP-siRNA抑制转染pEGFP-C1PC12细胞EGFP表达最为明显。Western-Blot显示50nM甲基化siRNA-Ⅰ、Ⅱ、Ⅲ可使PC12细胞EGR-1蛋白表达分别下降约85%、30%和78%;C6细胞EGR-1蛋白表达分别下降约90%、47%和85%。RT-PCR结果显示siRNA-Ⅰ、Ⅱ、Ⅲ分别下降PC12细胞Egr-1 mRNA数量的89%、47%和86%,C6细胞的92%、50%和85%。结论siRNA-Ⅰ和siRNA-Ⅲ可以充分下调细胞内Egr-1的表达,为实验需要的甲基化siRNA;FAM-siRNA和EGFP-siRNA可在检测转染效率的同时优化实验条件。
第三部分Egr-1 siRNA对吗啡戒断引起细胞凋亡的抑制作用及其机制研究
目的观察Egr-1 siRNA抑制吗啡戒断引起细胞凋亡的作用,探讨吗啡成瘾机制及治疗方法。
方法PC12细胞中加入100μmol/L吗啡, 48小时后给予10μmol/L纳络酮拮抗,利用倒置显微镜观测各组细胞形态及数量,然后EGR-1蛋白免疫荧光染色。接着转染50nM siRNAⅠ和siRNA-Ⅲ到PC12细胞,并设置N-siRNA-Ⅰ和N-siRNA-Ⅲ为对照以及阴性对照和阳性对照。同时慢性吗啡(100μmol/L)处理PC12细胞48小时,加入10μmol/L纳络酮,用MTT比色法观察PC12细胞的损害,应用FITC-AnnexinⅤ/PI流式凋亡检测试剂盒检测细胞凋亡率。
结果慢性吗啡处理组PC12细胞未见明显的荧光染色。吗啡慢性处理并急性戒断后1小时,即出现EGR-1蛋白阳性染色,但8小时后荧光染色细胞密度开始减少;24小时后光学显微镜观察细胞折光性能力减弱,细胞中颗粒增多,细胞皱缩,核固缩并断裂成数个,以及染色质凝集,大小不等的圆形颗粒及凋亡小体的形成。MTT法观察siRNA-Ⅰ和siRNA-Ⅲ能明显增加吗啡慢性处理急性戒断后细胞的存活率,差异有显著性意义(p<0.05);同时还观察到慢性吗啡处理组与空白对照组比较,生存率明显下降,差异有显著性意义(p<0.05);吗啡急性戒断24小时后,siRNA-Ⅰ和siRNA-Ⅲ分别能抑制27.7%和30.9%的细胞损害。与N-siRNA对照相比,流式细胞检测结果发现转染siRNA-Ⅰ和siRNA-Ⅲ的PC12细胞在吗啡急性戒断后,细胞凋亡水平分别下降了26.5%和20.3%。
结论Egr-1基因参与吗啡慢性处理和急性戒断后的细胞凋亡;下调Egr-1基因的表达,可降低吗啡戒断后细胞凋亡的发生率。
全文总结
1.EAAs、NMDA受体、GABAA受体、吗啡受体均参与调节Egr-1基因表达。
2.Egr-1基因参与吗啡慢性处理和急性戒断后的细胞凋亡。
3.下调Egr-1基因表达,可降低吗啡戒断后细胞凋亡的发生率。
4.Egr-1基因表达参与了阿片类药物的依赖和成瘾,具体机制可能与Egr-1基因表达增加诱发细胞凋亡有关。
Background
Chronic opiate applications lead to long-term impacts on many functions of the brain and induce tolerance, dependence and addiction. Addiction continues to exact enormous human and financial costs on society, but available treatments remain inadequate for most people. By analogy with other medical disorders, an improved understanding of the biological basis of addiction will lead to more effective treatments and eventually to cures and preventive measures. But it is regrettable that mechanisms of dependence and addiction are still not understood.
Previous research suggested that the abnormal gene expression in brain, impairment of neuronal cells and changes in the plasticity of neuron may play important roles in the development of opiate addiction. And chronic morphine treatment and morphine withdrawal cause apoptosis and injury of neuronal cells and other cells. So the neuronal injuries and impairments and changes in neuroplasticity in certain brain regions following chronic opiate treatment and opiate withdrawal could caused by apoptosis and have been implicated in the development of opioid dependence and addiction.
Egr-1, also called zif268, nerve growth factor-induced gene A (NGFI-A), belongs to the category of immediate early genes (IEG). It codes for a transcription factor protein, named EGR-1. Acting as a transcription factor, EGR-1 directly controls expression of other genes, which makes this protein an important object of studies aimed at understanding the orchestration of neuronal responses to a variety of stimuli. Specifically expressed in brain, Egr-1 can mediate cellular apoptosis and is involved in several drugs addiction. But it is unclear that Egr-1 is implicated in apoptosis resulted from chronic opiate treatment and opiate withdrawal, and the development of opioid addiction. The present study investigated the effect of acute morphine, glutamic acid, and midazolam administration, and chronic opiate treatment and opiate withdrawal on expression of Egr-1 in PC12 cells and C6 cells. Moreover, we design three Egr-1 siRNAs, and determine their functionality in PC12 cells and C6 cells. At last, selected potent Egr-1 siRNAs were transfected into PC12 cells. We study whether Egr-1siRNAs can inhibit cellular apoptosis and injuries following chronic opiate treatment and opiate withdrawal.
In this paper, the study was separated into three parts.
Part one The effect of different drugs on the expression of Egr-1. Aim To explore the effects of different drugs on levels of Egr-1 gene expression in PC12 cells or C6 cells.
Methods PC12 cells and Rat C6 glioma cells were seeded onto a 60-mm culture dish at a density of 1.5–2×106 cells/dish, and maintained in 5 ml of DMEM supplemented with 10% heat-inactivated fetal calf serum and 5% horse serum, 50 units/ml of penicillin, 50μg/ml of streptomycin at 37°C for 48 h in a humidified incubator containing 95% air–5% CO2 atmosphere. In the experiments testing whether glutamine acid, morphine, naloxone, and midazolam can induce EGR-1 protein expression, we treated the cells with 0-10000μM of glutamine acid, and 0-1000μM of morphine, naloxone, and midazolam. In the experiments testing the effects of pretreatment with morphine, naloxone, and midazolam were also present during treatment of the cells with glutamine acid. Immunocytochemical stain and western blot was used to detect the expression of Egr-1 gene.
Results EGR-1 protein staining were observed at 1-10000μM glutamine acid, and the optimal inducing concentration was 100μM. Furthermore, midazalam and morphine can induce expression of Egr-1 gene at a concentration more than 100μM and 1000μM respectively. Pretreatment of PC12 cells with 10μM midazolam, 100μM propofol, 100μM ketamine and 100μM morphine, the expression level of EGR-1 protein induced by glutamine acid significantly declined.
Conclusion Glutamine acid can induce expression of Egr-1 gene, but the level of Egr-1 gene expression induced by midazalam and morphine is determined by the concentrations of them. Moreover, midazolam, propofol, ketamine and morphine also could inhibit glutamine acid induced -EGR-1 protein expression.
Part two Inhibition of Egr-1 gene expression by Egr-1 siRNA. Aim To design Egr-1 siRNAs for selecting potent siRNAs and facilitating functional gene knockdown studies.
Methods Three Egr-1 siRNAs were designed and synthesized, siRNA-Ⅰ: sense, (5'-3') CCAACAGUGGCAACACUUUdTdT, antisense, (3'-5')dTdTGGUUGUCAC -CGU UG UGAAA; siRNA-Ⅱ:sense, (5'-3') GACUUAAAGGCUCUUAAUA dTdT; antisense, (3'-5')dTdT CUGAAUUUCCGAGAAUUAU; siRNA-Ⅲ: sense, (5'-3') GGAC AAGAAA GCAGACAAA dTdT, antisense, (3'-5')dTdTCCUGUUCUUUCGU–CUGUUU. In order to enhance serum and intracellular stability of siRNA, end modification of three terminal by chemical modifications were performed, such as 2’-OMe substitutions. Transfections of different concentrations of FAM-siRNA were performed with Lipofectamine TM 2000.
Threir transfection efficiency were determined by immunocytochemistry. In addition, 0-100μM EGFP–siRNAs were used to optimized transfection and test procedures in 6-well plates. Following transfection of three designed siRNA into PC12 cells and C6 cells at a concentration of 50nM, the level of glutamine acid induced-Egr-1 mRNA and EGR-1 protein were observed 48 later by RT-PCR and western-blot respectively.
Results Transfection efficiencies of FAM- siRNAs were 80%-90% at a concentration of 50nM, and increasing the concentration did not improve transfection effiencies. Two hours after transfection of EGFP–siRNAs into PC12 cells with pEGFP-C1, the suppressing expression of green fluorescence protein was observed. In addition, EGFP–siRNA had the most potent efficiency of inhibition at 50nM. 48 houes after siRNAⅠ,Ⅱ,Ⅲwere deliveried into PC12 cells and C6 cells, the levels of Egr-1 mRNA and EGR-1 protein induced by glutamine acid had reduced 89%, 47%, 86% and 85%, 30%, 78% in PC12 cells repectively. And in C6 cells, the reducing levels were 92%, 50%, 85% and 90%, 47%, 85% repectively.
Conclusion siRNAⅠ,Ⅲare able to down-regulate the expression of Egr-1 gene, and can be used to facilitate functional gene knockdown studies. In addition, EGFP-siRNA and FAM- siRNA can be used to determine the transfection efficiencies and optimized experimental procedures.
Part three The effect of Egr-1 siRNAs on cellular injuries and apoptosis following morphine withdrawal.
Aim To study the effect of Egr-1 siRNAs on cellular injuries and apoptosis following opiate withdrawal, and explore the mechanism of morphine addiction.
Methods After the cells were treated with 100μmol/ L morphine for 48 h, 10μmol/ L naloxone was used to produce acute withdrawal. The invert microscope was applied to observe morphology of the cells, and EGR-1 protein was detected by immunocytochemistry. Following transfection of siRNA, PC12 cells were treated with morphine for 48 h. After acute withdrawal with naloxone, thiazolyl blue tetrazolium bromide (MTT) assays were used to detect the proliferation of the cells and cells apoptosis were determined by AnnexinⅤ/PI flow cytometry.
Results Under invert microscope, the forms of cells were irregular and spindle, the chromatin concentrated into masses, the apoptosis bodies were formed 24h after morphine withdral. EGR-1 protein expression was appeared at 1h after withdrawal, reached peak at 8h and was detected even at 24h. MTT assays founded that mortality declined significantly (p<0.05 ), and apoptosis of PC12 cells who transfected siRNAⅠ,Ⅲreduced 26.5% and 20.3% respectively.
Conclusion Egr-1 gene is involved in apoptosis after chronic morphine treatment and acute morphine withidrawal. Down-regluation of Egr-1 gene can reduce apoptosis of cells induced by opiate withdrawal.
Summary
1. Glutamine acid could induce expression of Egr-1 gene, and midazalam, morphine, propofol and ketamine could inhibit its expression.
2. The designed siRNAⅠ,Ⅲcan down-regulate the expression of Egr-1 gene in PC12cells and C6 cells.
3. Egr-1 gene is involved in apoptosis after chronic morphine treatment and morphine withidrawal.
4. Down-regulation of Egr-1 gene can reduce apoptosis of cells induced by morphine withdrawal.
5. Apoptosis following chronic opiate treatment and opiate withdrawal could be caused by Egr-1 gene and could be implicated in the development of opioid dependence and addiction.
引文
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