CN114214396A - Gabrd甲基化作为抗***复吸靶点的应用 - Google Patents
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Abstract
本发明涉及一种GABRD甲基化作为抗***复吸靶点的应用,与现有技术相比,本发明的优点在于:本发明通过研究发现降低GABRD基因甲基化水平能够有效减少***复吸行为,说明以GABRD基因甲基化作为靶点治疗***复吸的药物具有重要的研发价值和开发意义。
Description
技术领域
本发明涉及一种GABRD甲基化靶点,尤其涉及一种以GABRD甲基化作为抗***复吸靶点的应用。
背景技术
阿片类药物成瘾尤其是***成瘾导致巨大的医疗和经济危害。***毒品和犯罪问题办公室(UNODC)调查数据显示,在全球范围内,2018年全年破获毒品犯罪案件10.96万起,抓获犯罪嫌疑人13.74万名,缴获各类毒品67.9吨,***成瘾人群仍然占取较大的比例,***滥用形势严峻。***长期激活阿片受体使阿片受体及非阿片受体***发生代偿性适应,从而导致成瘾。长期慢性使用***出现强烈的躯体依赖和精神依赖,由此产生对再次用药的强烈渴求,这种渴求是导致复吸率居高不下的主要原因。中脑腹侧被盖(VTA)到伏隔核(NAc)脑区多巴胺能***是目前公认的药物奖赏的主要通路。NAc是大脑奖赏回路中的重要核团,伏隔核中的中型多棘神经元,会产生一种主要的中枢神经***的抑制性神经递质γ-氨基丁酸(GABA)。其中A型GABA受体δ亚型(GABRD)基因被证明与癫痫、惊厥等疾病有关,并发现该基因的甲基化可能参与***等药物的成瘾和复吸。因此对***成瘾干预机制的研究和药物治疗靶点的筛选,有利于促进***成瘾和复吸的预防与治疗。
发明内容
本发明所要解决的技术问题是针对上述现有技术现状而提供一种以GABRD甲基化作为抗***复吸靶点的应用。
本发明解决上述技术问题所采用的技术方案为:一种以GABRD甲基化作为药物靶标在筛选***戒断后预防复吸的药物中的应用,其特征在于:GABRD的差异甲基化区域DMR序列为:
CGGACAGCACCCGGGAGGCGGGAGAGAGGTCAGAGGGGTCCTCAGAACTGGAATAGCAGCACGCG。
进一步地,扩增GABRD的差异甲基化区域DMR序列的扩增引物和测序引物分别为:
甲基化特异性上游引物:5’-Biotin-GTGGTTAAGGGTAAGAATGAAGAGA-3’;
甲基化特异性下游引物,5’-CCCATTAAATCCACCCAATTTTACTT-3’;
甲基化特异性测序引物,5’-CCACCCAATTTTACTTT-3’。
进一步地,所述预防复吸的药物为GABRD甲基化转移酶抑制剂。
5’AZA是DNA甲基转移酶抑制剂,该药对GABRD甲基化具有抑制作用,本发明经外周注射5’AZA对***自身给药大鼠消退后复吸行为的影响、中枢注射5’AZA对***自身给药大鼠消退后复吸行为的影响,评价5’AZA对***戒断后预防复吸的抑制作用效果;并通过中枢注射5’AZA对***自身给药消退后复吸大鼠NAc脑区GABRD基因DNA甲基化及蛋白表达的变化,观察GABRD甲基化作为戒毒治疗靶点的可能性。
结果可见:外周注射5’AZA能有效抑制***觅药行为的恢复、中枢注射5’AZA能有效抑制***觅药行为的恢复,以上结果表明:GABRD甲基化在药物成瘾中起着重要作用,针对其设计药物有望戒除毒瘾。
与现有技术相比,本发明的优点在于:本发明通过研究发现降低GABRD基因甲基化水平能够有效减少***复吸行为,说明以GABRD基因甲基化作为靶点治疗***复吸的药物具有重要的研发价值和开发意义。
附图说明
图1A为本发明的***模型构建中有效鼻触和无效鼻触数记录(***自身给药组Heroin组;生理盐水被动给药组Control组);
图1B为本发明的***模型构建中注射针数记录(***自身给药组Heroinself-administration组;***被动给药组Yoke heroin组;生理盐水被动给药组Control组);
图2为本发明的GABRD候选片段及待测位点位置信息;
图3为本发明的GABRD基因甲基化在***自身给药组(Heroin self-administration组),***被动给药组(Yoke heroin组)和对照组(Control组)中的变化情况;
图4为本发明的GABRD基因中CG3测试位点在***自身给药组中甲基化水平显著降低的结果图;
图5为本发明的GABRD基因中CG4测试位点在***自身给药组中甲基化水平显著降低的结果图;
图6为本发明的GABRD基因中CG5测试位点在***自身给药组中甲基化水平显著降低的结果图;
图7为本发明的GABRD在***自身给药组(Heroin self-administration组),***被动给药组(Yoke heroin组)和对照组(Control组)中mRNA表达情况;
图8为本发明的GABRD在***自身给药组(Heroin self-administration组),***被动给药组(Yoke heroin组)和对照组(Control组)蛋白表达情况;
图9为GABRD基因候选片段以及片段甲基化功能验证结果图(Pro即pGL3-promoter质粒,F-luc:Firefly luciferase萤火虫荧光素酶;R-Luc:Renilla luciferase海肾荧光素酶;Pro:pGL3-promoter空载对照组;Pro-GABRD:GABRD***组pGL3-promoter-GABRD;Me-Pro-GABRD:甲基化处理的GABRD***组即pGL3-promoter-GABRD甲基化处理组;
图10为本发明的外周注射甲基化转移酶抑制剂5’AZA对大鼠***自身给药消退后***引燃的复吸行为的影响(Active:有效鼻触反应数;Inactive:无效鼻触反应数;Heroin:***;SAL:生理盐水;5’AZA:5-氮杂-2'-脱氧胞嘧啶核苷,5-aza-2-deoxycytidine);
图11为本发明的外周注射甲基化转移酶抑制剂5’AZA对大鼠条***自身给药消退后条件性线索诱导复吸行为的影响(Active:有效鼻触反应数;Inactive:无效鼻触反应数;Heroin:***;SAL:生理盐水;5’AZA:5-氮杂-2'-脱氧胞嘧啶核苷,5-aza-2-deoxycytidine);
图12为本发明的NAc脑区微注射甲基化转移酶抑制剂5’AZA对大鼠***引燃的复吸行为的影响(Active:有效鼻触反应数;Inactive:无效鼻触反应数;Her:***,Heroin;Sal:生理盐水,Saline;aCSF:溶媒剂;5’AZA:5-氮杂-2'-脱氧胞嘧啶核苷,5-aza-2-deoxycytidine);
图13为本发明的NAc脑区微注射甲基化转移酶抑制剂5’AZA对NAc脑区GABRD基因甲基化的影响(Her:***,Heroin;Sal:生理盐水,Saline;aCSF:溶媒剂;5’AZA:5-氮杂-2'-脱氧胞嘧啶核苷,5-aza-2-deoxycytidine);
图14为本发明的NAc脑区注射甲基化转移酶抑制剂5’AZA对GABRD蛋白表达的影响(Her:***,Heroin;Sal:生理盐水,Saline;aCSF:溶媒剂;5’AZA:5-氮杂-2'-脱氧胞嘧啶核苷,5-aza-2-deoxycytidine)。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。
本发明实施例中使用的***纯度为98%,由公安部物证鉴定中心提供。
实施例1***静脉自身给药模型的建立
静脉自身给药(Intravenous Drug Self-Administration)是研究药物成瘾最经典的动物模型之一,静脉自身给药模型是反映用药者主动觅药和用药行为的经典动物模型,可以利用实验动物考察给药动机和主动强迫性用药行为。
经典的大鼠***自身给药模型可以很好的模拟临床上吸毒患者的***吸食行为,大鼠经过训练可以产生对***的自我给药,同时训练过程中不断上升的对***的行为反应率和给药量可以反映吸毒患者从一开始接触***到大量吸食***成瘾的强迫性觅药行为。
我们采用如下方法来建立大鼠的自身给药模型:首先,SD雄性大鼠进行颈静脉插管手术,在右颈静脉***一段PE管,并经背部穿出体外,手术后抗菌和恢复一周以上。随后,采用与外界隔音隔光的自身给药笼,笼内装有鼻触灯用来记录有效鼻触和无效鼻触,顶部笼灯连接注射泵,形成鼻触灯、笼灯和注射泵一体化装置。自身给药训练前,将注射泵上的注射器装满5mL***溶液,待大鼠置于笼中后将笼上连接管与大鼠背部插管连接。训练程序为FR-1强化训练,开启程序,此时有效鼻触内的黄色信号灯亮起,大鼠触碰有效鼻触后,信号灯熄灭而笼灯亮起,并伴随着注射泵的蠕动声获得一针***,笼灯每次持续亮5s,***剂量为:第1-2天为0.1mg/kg,第3-6天为0.05mg/kg,第7-14天为0.025mg/kg。在之后的20s内,有效鼻触处于不应期(Time out),即大鼠在这期间触碰有效鼻触仅在电脑上记录数据,但并不能真正得到***,不应期后进入下一个循环,在***自身给药大鼠4h训练的同时,被动***给药组大鼠也给予注射等量的***,而被动生理盐水组大鼠同时注射同等剂量的生理盐水作为正常对照组。每天进行1次4h的自身给药训练,连续14天,有效触鼻反应率和注射量均明显上升,且最后3天有效鼻触数上下浮动不超过10%(见图1A)。从图1B可以看出,经过14天的自身给药训练,***自身给药组和被动给药组大鼠***注射针数显著高于生理盐水组,并维持在40针以上,表明已成功建立稳定的***自身给药模型,符合后续实验的要求。
实施例2Illumina HiSeq测序
于稳定建立***自身给药模型12h后,大鼠用戊巴比妥钠腹膜内给药,深度麻醉后处死分离NAc脑区。设立***自身给药组(Heroin self-administration),***被动给药组(Yoke heroin组)和被动生理盐水对照组(Control)(每组n=3)。应用PerkinElmer全自动核酸提取仪(珀金埃尔默医学诊断产品上海有限公司)提取组织样本的基因组DNA,再通过Qubit4.0核酸蛋白测定仪检测所得DNA的浓度。DNA片段化及纯化后进行末端修复并添加测序接头,然后进行Bisulfite处理,扩增后,进行文库构建及质检,然后上机进行Illumina HiSeq全基因组甲基化测序。最后分析筛选差异甲基化位点及区域(Differentmethylation region,DMR),比较各组甲基化水平变化,筛选发现GABRD基因上存在DMR,位于chr5:172809066-172809129区域同时位于CpG52岛上(见表I及图2)。
而DMR序列为:
CGGACAGCACCCGGGAGGCGGGAGAGAGGTCAGAGGGGTCCTCAGAACTGGAATAGCAGCACGCG;
表I:测序检测GABRD基因DMR区域信息
>rn6_cpgIslandExt_CpG:52 range=chr5:172809013-172809608 5'pad=0 3'pad=0 strand=+repeatMasking=none
CGCCTGGCGACTCCAATCCCTCCCCAAAGCTCAGCGTTCAGCACCGAGGACGGCGGACAGCACCCGGGAGGCGGGAGAGAGGTCAGAGGGGTCCTCAGAACTGGAATAGCAGCACGCGCAAAGCAAAACTGGGTGGACCTAATGGGTGAAGACTCGGGGTTCTGGCAACTCGGAACTCTGCCCCACCGAGGGTGCCCGCCAAAGGACGAGTCCCTCCCGCCTCATATCGTCCCCGGCCCAGGAGCTGCTCACCTGGCGCCATGGTGCGGCTGCGTGCACAGCAGAAGGAGCGGCAGCAGCAGCCAGCCCAGAACGTCCATGGCCTTGCGGGCTGTGCGCGGGACAGGTCCGGCTAGCCCCAGCGCAAGCAAAGTTGCTTCGCCTGCGGCCGCGCGGCGGCACCAGAAGGGCGGGAGCGCGAAGGAGAGTGGAACGGGCGCGGGGGGAGGACGGAACGTCGAGCCTGCCCCGCCCCGCCCCGCCCGGCGCTCCAGTCCCACCCCCGCTCGCGCGCCCCACTGCACGCGGGAGGCTTCTGCGCACCGCATAGCACCGGGTGGTAGCTCCACCGCGCTCCCAGAGCCTCGCACACCGCG
同时可对应地参见序列表的SEQ ID NO.1。
实施例3扩大样本检测GABRD基因DNA甲基化水平变化
按照实施例1重新建立大鼠***自身给药模型,设立***自身给药组(Heroinself-administration),***被动给药组(Yoke heroin组)和被动生理盐水对照组(Control)。取各组NAc脑组织,提取组织DNA。采用重亚硫酸盐焦磷酸测序技术对GABRD基因CpG岛区域的DMR中5个CpG位点进行了DNA甲基化水平检测。此技术的基本原理:用试剂盒中的重亚硫酸盐处理DNA样品后,再应用聚合酶链式反应(PCR)用甲基化特异性扩增上下游引物扩增,然后通过特异性测序引物对扩增产物进行焦磷酸测序,从而计算得到各个位点甲基化程度。本次研究采用PyroMark Assay Design 2.0软件进行引物设计,用于实验的PCR扩增引物和测序引物如下:
(1)甲基化特异性上游引物(Forward primer)
5’-Biotin-GTGGTTAAGGGTAAGAATGAAGAGA-3’;
(2)甲基化特异性下游引物(Reverse primer)
5’-CCCATTAAATCCACCCAATTTTACTT-3’;
(3)甲基化特异性测序引物(Sequencing primer)
5’-CCACCCAATTTTACTTT-3’。
上述扩增的具体步骤为:
a、采用使用Qiagen EpiTect Bisulfite Kit试剂盒(Qiagen,#59104)对成瘾及对照者基因组DNA进行亚硫酸氢盐转化。
b、取步骤A中转化后的DNA样本10-20ng加入到Pyromark PCR试剂盒(PyromarkPCR Kit;Qiagen;#978703),并加入上述一对GABRD基因CpG52区域甲基化特异性扩增引物,进行PCR扩增,扩增条件:首先95℃3min的变性;接着95℃30s,60℃30s,72℃1min,共40个循环的反应;然后延伸反应72℃7min。
c、Pyrosequencing检测,在PyroMark Q48软件中设计好需要运行的程序,将程序导入U盘中,插到PyroMark Q48的USB接口中。点击仪器触屏上显示的“Sequence”,载入U盘中的运行程序。放入吸废液的棉条,保证首尾相接处为正左(9点钟)方向。推开仪器的盖子,打开每个卡夹的盖子,选择需要灌注的卡夹,选中后的卡夹会显示水滴图标,选择本次run是否是最后一个run。根据触屏上显示的每种试剂的体积,将试剂依次加入到各个卡夹中。将试剂加入卡夹时,请使用加样枪贴壁缓慢加入,避免产生气泡。加入试剂后,盖上卡夹的盖子并且上锁。点击“Start”开始测试卡夹功能是否正常,测试通过的卡夹后面会出现绿色的箭头。将上样圆盘放入机器中,注意对准圆盘上确定位置的孔,否则机器将会报警。拧上固定上样圆盘的固定螺丝。振荡磁珠,然后使用随机器送的连续加样枪加磁珠到上样圆盘,每孔3μl,可一次性连续加3个孔。在上样圆盘的孔中加入10μl含生物素标记的PCR产物,如PCR产物不够10μl,要用纯水补到10μl,确保PCR产物液体完全覆盖住了磁珠。运行Q48程序,点击“Start”。运行结束,结果会自动导入U盘,拔掉U盘,先从软件上关闭仪器,再关闭仪器。最后,使用焦磷酸测序仪自带的Pyro Q CpG软件分析每个位点甲基化状态。从焦磷酸测序仪中得到的分析的结果数据(见图3示例)。横轴为加入的dDNTP顺序,纵轴代表荧光信号强度。其中蓝色的测序质量最好,黄色其次,红色的质量最差。但如果一个反应中有蓝色的,就说明整体质量是没有问题的,红色的和黄色的结果也正常。采用SPSS 16.0对数据进行分析发现:被检测的5个位点(CpG1、CpG2、CpG3、CpG4和CpG5)之间存在显著相关性(R>0.5,P<0.01,见图2表格内R值),因此可对CpG1-CpG5总体甲基化进行分析。发现***自身给药组,被动给药组与对照组间GABRD基因CpG52区域甲基化水平的差异,结果(见图3发现相比于对照组,***自身给药组GABRD甲基化水平显著降低(P=0.012)。各个位点分别比较分析,显示相对于对照组,***自身给药组中CG3、CG4、CG5三个位点甲基化水平均显著性降低(见图4、5、6)。
实施例4 NAc脑区GABRD基因mRNA表达水平变化
通过Roche LightCycler 480仪反转录实时定量PCR(RT-qPCR)检测GBRD基因mRNA表达情况。提取***自身给药组,被动给药组和对照组大鼠NAc脑组织RNA,总RNA使用(Invitrogen;Thermo Fisher Scientific,Inc.)试剂盒提取。使用miScript IIRT试剂盒(Qiagen gmbH),把总RNA反转录合成cDNA。qPCR引物核苷酸序列显示在表II,GAPDH基因作为内参。反应体系包含有5μl SYBR Green预混物,2μl引物(10μM),1.5μl cDNA和1.5μl水。扩增条件为:95℃,10min,40个循环:95℃,10S,55℃,15S和72℃,15S。使用2-ΔΔCq方法分析RT-qPCR数据分析GABRD mRNA表达水平的相对变化。结果分析显示,分别相对于对照组与***被动给药组,***自身给药组GABRD基因mRNA表达水平均显著升高(F(3,24)=9.065,P=3.42E-4;F(2,24)=10.238,P=0.001,见图7)。
表II qPCR引物序列信息
实施例5 NAc脑区GABRD基因蛋白表达水平变化
同时利用Western Blot方法检测***自身给药组(Heroin self-administration),***被动给药组(Yoke heroin组)和被动生理盐水对照组(Control)中NAc脑区GABRD蛋白表达水平。检测方法如下:融解RIPA裂解液(碧云天,P0013B),混匀。取适当量的裂解液,在使用前数分钟内加入PMSF,使PMSF的最终浓度为1mM,组织中加入150μl裂解液,使用匀浆器匀浆,然后用移液器反复吹打,破碎组织裂解充分。置于冰上放置30min,每隔10min涡旋一次,使组织裂解充分,4℃10000-14000g离心10min,取上清,即可进行后续的蛋白电泳操作;将蛋白液与loading buffer一起混匀后,放入100℃水浴5min,离心,放置冰上,待上样。配制SDS-PAGE分离胶,加入两块制胶玻璃板中,用异丙醇封平。待下层胶凝固后,将异丙醇倒掉,用ddH2O冲洗后,吸干,开始配制浓缩胶。将浓缩胶加入两块制胶玻璃板中,不要有气泡,立即将梳子***,凝固后将梳子拔出。做好制胶槽后,放在冰上,将电泳液倒入后,开始上样,每个孔上样10-35μl。记录上样样本顺序,事先配制Runningbuffer(Tris 3.028g(25mM),Gly 14.413g(192mM),10%SDS 10mL(0.1%SDS),用ddH2O定容至1000mL,需放在4℃预冷。Transfer buffer(Tris 3.028g)(25mM),Gly 14.413g(192mM),甲醇(Methanol)200mL用ddH2O定容至1000mL),需放在4℃预冷。上层胶60v,30min,下层胶110v,120min。电泳结束后需要将胶放在PBS润洗2min,再放入转膜缓冲液中浸泡平衡。将剪好的PVDF膜先在甲醇中浸泡2min,后在ddH2O中浸泡2min,最后在transferbuffer中浸泡平衡15min。垫子、膜和胶的放置:红、(+)白架子、垫子、滤纸、膜、胶、滤纸、垫子、黑架子(-)、黑。转膜条带是从-到+。冰上转移,100v,40min/70min。转移结束后将膜放入PBS中浸泡3-5min,剪膜。用5%脱脂牛奶(0.75g奶粉+15mL PBS)在37℃封闭1-2h。采用1×PBS-T(1000mL 1×PBS+1mL Tween-20)5-10min,洗膜三次。孵育一抗,内参(1:5000),GABRD蛋白(均按1:1000稀释),4℃,摇床,过夜(冷库)。从冰库中取出摇床,洗膜,1×PBST 5-10min,三次。加二抗(1:10000),37℃,2h,摇床,用1×PBST 5min,洗膜五次。化学发光显影冰记录保存结果,进行灰度分析。结果显示,组间蛋白表达水平存在显著差异(F(2,3)=21.942,P=0.016),多重比较显示分别相对于对照组与***被动给药组,***自身给药组GABRD基因mRNA表达水平均显著升高(P=0.016,P=0.009,见图8)。
实施例6双荧光素酶报告基因实验
双荧光素酶报告基因载体***含有可在同一细胞中同时表达的两种荧光素酶基因,包括萤火虫荧光素酶报告基因和海肾荧光素酶报告基因。将所研究目的基因的调控序列克隆到含有报告基因的表达质粒中,然后将重组质粒导入适当的细胞系,通过测定报告基因表达的水平来间接评价目的序列对基因表达的诱导作用。
本发明为了验证GABRD基因目的序列的生物学功能,检验目的序列是否有增强子活性,并检测序列甲基化对其活性的影响,开展了双荧光素酶报告基因实验,首先进行293T细胞扩增培养,然后合成大鼠GABRD目的DMR序列:CGGACAGCACCCGGGAGGCGGGAGAGAGGTCAGAGGGGTCCTCAGAACTGGAATAGCAGCACGCG,将合成的序列***到PGL3-Promoter构建质粒。对构建好的产物使用SssI试剂盒(Thermo,EM0821)处理进行体外甲基化。使用EpiJET DNAMethylation Analysis Kit进行甲基化鉴定,电泳验证甲基化效果,随后将构建成功及甲基化成功的质粒转染293T细胞,48h后进行双荧光素酶报告基因检测,分组如下:pGL3-promoter空载对照组(Pro);GABRD***组pGL3-promoter-GABRD(Pro-GABRD);甲基化处理的GABRD***组即pGL3-promoter-GABRD甲基化处理组(Me-Pro-GABRD)。检测前弃去培养基润洗后,每孔加100μl稀释好的1x PLB,摇床常温条件下摇15min,裂解细胞。将裂解液收集至1.5mL的Ep管中,10000g离心5min,取上清,备用。打开多功能酶标仪(TECAN InfiniteM1000 Pro)),打开软件,将10mL预先混好的荧光素酶测试试剂LAR II加入到15mL的离心管中,放入A通道吸管;将200μl 50XSubstrate加入到10mLBuffer中,配置成Stop&Glo Reagent,再将Stop&Glo Reagent加入到15mL的离心管中,放入B通道吸管,打开软件,设置好参数。白色不透光的96孔酶标板中每孔加入备用的上清液50μl,放入多功能酶标仪中,开始检测,每孔先加入50μl预先混好的荧光素酶测试试剂LARII,2s后测数据,再每孔添加50μl Stop&Glo Reagent,静止2s后,测数据,根据得到的比值来比较不同样品间目的报告基因的激活程度;然后使用Graphpad prism(Graphpad Software,SanDiego,CA)软件分析,统计方法为One-way anova检验,随后SNK检验组间差异,以P<0.05为显著性差异的筛选标准。如图9的结果显示,与Pro组相比,Pro-GABRD组荧光比值显著升高,甲基化处理组Me-Pro-GABRD荧光比值显著降低,结果提示GABRD基因DMR序列有显著的增强子样的功能,且片段经甲基化处理后其类增强子样活性被显著抑制。结果表明,GABRD目的DMR序列有增强子的作用,而GABRD基因甲基化处理后降低其基因表达启动功能。
实施例7 5’AZA对***自身给药大鼠消退后条件性线索诱导或***引燃的复吸行为的影响
复吸是临床上治疗***成瘾的难点,***患者进行脱毒戒断以后,再次接触以往吸毒相关环境、毒品本身或者毒友唆使诱导下,往往会产生吸食***的冲动,并最终导致***复吸。大鼠的复吸模型能够较好地反映处于***戒断期的患者再次接触以往吸食***相关的环境或者***本身后,导致***复吸(再次成瘾)的行为。
(1)外周注射5’AZA对***自身给药大鼠消退后复吸行为的影响
为了评价5’AZA是否对***成瘾大鼠的复吸行为有干预作用,我们分别建立了条件性线索或***引燃诱导的大鼠复吸模型,并进行复吸行为的测试。首先,训练大鼠在自身给药操作笼内经过14天的***自身给药训练,采用FR1程序(4h/days),所有实验大鼠进行9天的***或生理盐水自身给药训练,然后,在第10天,将***自身给药的大鼠随机分成***+5-Aza-dc给药组(Heroin/5’AZA)和***+生理盐水组(Heroin/SAL),Heroin/5’AZA组大鼠在每天***自身给药训练前半小时给予5’AZA(5mg/kg,s.c.),Heroin/SAL组大鼠在每天自身给药训练前半小时给予生理盐水代替5’AZA处理,连续训练8天。统计学结果分析显示,外周注射DNA甲基转移酶抑制剂5’AZA可显著降低自身给药大鼠***引燃的复吸有效鼻触反应数(F(2,6)=9.249,P<0.05,见图10),而外周注射5’AZA对***自身给药大鼠条件性线索诱导的复吸有效鼻触数有降低趋势,但无统计学意义(F(2,6)=0.012,P=0.915,见图11)。结果提示外周注射5’AZA能有效抑制***觅药行为的恢复。
(2)中枢注射5’AZA对***自身给药大鼠消退后复吸行为的影响
为了进一步评价5’AZA对***自身给药大鼠复吸行为的影响是否通过中枢发挥作用的,我们重新建立了条件性线索或***引燃诱导的大鼠复吸模型,并进行复吸行为的测试。首先,所有实验大鼠采用FR1程序(4h/days)进行14天的***或生理盐水自身给药训练,然后进行消退训练,在消退训练的第10天开始,在训练前半小时在NAc脑区隔天双侧微注射5’AZA(1ug/侧,浓度1mg/ml)或aCSF,共消退14天,将***和生理盐水自身给药的大鼠随机分成***+5-Aza-dc给药组(Her/5’AZA)、***+生理盐水组(Her/aCSF)、生理盐水+5-Aza-dc给药组(Sal/5’AZA)和生理盐水+溶媒剂(Sal/aCSF)。统计学分析结果显示,5-Aza-dC(F(1,19)=14.402,P=0.001),heroin(F(1,19)=28.475,P=5.46E-5)以及交互作用(heroin和5-Aza-dC)(F(1,19)=16.367,P=8.40E-4)均可作为主效应导致有效鼻触反应数发生改变,多重比较显示相较于Heroin/aCSF组,Heroin/5’AZA组的***引燃的复吸有效鼻触反应数显著降低(P=2.45E-5,见图12)。结果提示中枢注射5’AZA能有效抑制***觅药行为的恢复。
实施例8中枢注射5’AZA对***自身给药消退后复吸大鼠NAc脑区GABRD基因DNA甲基化及蛋白表达的影响
对实施例7中的所有大鼠经复吸行为测试后立即处死,取NAc脑区,分别提取DNA和蛋白,DNA用焦磷酸测序方法,检测GABRD基因目的片段的甲基化水平。结果显示,相较于Heroin/aCSF组,Heroin/5’AZA组GABRD基因目的片段甲基化水平显著降低(结果如图13:44.71±1.19,32.68±5.29,P=0.038)。同时相较于Sal/aCSF,Sal/5’AZA组GABRD基因目的片段甲基化水平也显著降低(36.99±1.17,11.36±2.56,P=4.64E-4)。此外,Heroin/SAL相对于Control/SAL组甲基化有升高的趋势,但是无统计学差异,具体见下表:
另外对提取的蛋白用WB进行蛋白表达水平的检测,结果发现相较于Heroin/aCSF组,Heroin/5’AZA组GABRD蛋白表达水平显著升高(结果如图14:P=0.018)。同时相较于Sal/aCSF,Sal/5’AZA组GABRD基因目的片段甲基化水平也显著升高(P=0.010)。结果提示,中枢注射5’AZA有效抑制***觅药行为的恢复,可能部分是通过降低GABRD基因的甲基化增加其蛋白表达水平而发挥作用。
序列表
<110> 宁波市康宁医院(宁波市精神疾病预防控制中心、宁波市微循环与莨菪类药研究所)
<120> GABRD甲基化作为抗***复吸靶点的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 596
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cgcctggcga ctccaatccc tccccaaagc tcagcgttca gcaccgagga cggcggacag 60
cacccgggag gcgggagaga ggtcagaggg gtcctcagaa ctggaatagc agcacgcgca 120
aagcaaaact gggtggacct aatgggtgaa gactcggggt tctggcaact cggaactctg 180
ccccaccgag ggtgcccgcc aaaggacgag tccctcccgc ctcatatcgt ccccggccca 240
ggagctgctc acctggcgcc atggtgcggc tgcgtgcaca gcagaaggag cggcagcagc 300
agccagccca gaacgtccat ggccttgcgg gctgtgcgcg ggacaggtcc ggctagcccc 360
agcgcaagca aagttgcttc gcctgcggcc gcgcggcggc accagaaggg cgggagcgcg 420
aaggagagtg gaacgggcgc ggggggagga cggaacgtcg agcctgcccc gccccgcccc 480
gcccggcgct ccagtcccac ccccgctcgc gcgccccact gcacgcggga ggcttctgcg 540
caccgcatag caccgggtgg tagctccacc gcgctcccag agcctcgcac accgcg 596
Claims (3)
1.一种以GABRD甲基化作为药物靶标在筛选***戒断后预防复吸的药物中的应用,其特征在于:GABRD的差异甲基化区域DMR序列为:
CGGACAGCACCCGGGAGGCGGGAGAGAGGTCAGAGGGGTCCTCAGAACTGGAATAGCAGCACGCG。
2.根据权利要求1所述的应用,其特征在于:扩增GABRD的差异甲基化区域DMR序列的扩增引物和测序引物分别为:
甲基化特异性上游引物:5’-Biotin-GTGGTTAAGGGTAAGAATGAAGAGA-3’;
甲基化特异性下游引物,5’-CCCATTAAATCCACCCAATTTTACTT-3’;
甲基化特异性测序引物,5’-CCACCCAATTTTACTTT-3’。
3.根据权利要求1所述的应用,其特征在于:所述预防复吸的药物为GABRD甲基化转移酶抑制剂。
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