CN117604116A - 组蛋白甲基转移酶setd2作为神经病理性疼痛药物靶点的应用 - Google Patents
组蛋白甲基转移酶setd2作为神经病理性疼痛药物靶点的应用 Download PDFInfo
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Abstract
本发明公开了组蛋白甲基转移酶SETD2作为神经病理性疼痛药物靶点的应用。本发明公开了SETD2通过调节NMDA受体表达从而影响外周神经元兴奋性,改变了机体对疼痛信号的敏感性,因此可能成为诊断以及治疗疼痛症状疾病的靶点。本发明提供了新的疼痛症状疾病诊断及治疗的靶点,阐明了病理性疼痛的新机制,使用的药物较目前临床在用的阿片类药物、非甾体类抗炎药、抗抑郁药物等,药物依赖与药物不良反应更低,整体安全性更高,针对性更强。
Description
技术领域
本发明涉及组蛋白甲基转移酶SETD2作为神经病理性疼痛药物靶点的应用。属于医药技术领域。
背景技术
慢性神经病理性疼痛是临床上常见的疾病并发症,通常由周围神经损伤、带状疱疹后神经痛、糖尿病神经病变和化疗引起,是一难治的临床问题,也是世界性的公共健康问题。流行病学观察显示,全球约有6.9~10%的人口患有或曾患神经病理性疼痛。慢性、持续的疼痛可能是患者就医最常见的原因,目前以药物治疗为主,但治疗效果不甚理想。临床可选药物主要包括阿片类药物、非甾体类抗炎药、抗抑郁药物等,但长时间用药治疗过程中往往会出现一定的药物依赖和药物不良反应,如便秘、恶心、呕吐等,停药后病情易复发,整体安全性与有效性仍受争议。
神经病理性疼痛形成主要包括中枢、外周神经系统病理条件下的适应性结构改变、细胞间相互作用以及疼痛信号通路敏化。其中参与的生物进程有离子通道的改变,免疫细胞的激活,神经胶质细胞分泌的神经递质和表观遗传的调控。外周炎症和神经损伤使背根神经节(DRG)、脊髓和大脑疼痛相关区域的初级感觉神经元中的受体、酶、离子通道、神经递质、神经调节物质和结构蛋白的表达发生转录和翻译变化。研究表明,创伤后这些基因会因表观遗传酶的失调而改变启动子的状态。包括甲基转移酶和脱甲基酶,以及组蛋白去乙酰化酶和组蛋白乙酰化酶等。因此,表观遗传酶引发了化学介质启动子转录的可及性,从而影响基因表达,改变这些酶的表达或活性可以减少炎症的产生以及减轻神经性疼痛。已有研究清楚地表明,神经系统的损伤会引发启动子甲基化的增加,产生基因沉默。神经损伤引起甲基转移酶如EHMT2或G9a、DNA甲基转移酶3α(DNMT3a)和神经元抑制沉默因子(NRSF)的上调,这些过度表达的表观遗传酶对类似目标基因的沉默有明显的影响,如钾离子通道、钠离子通道等,与疼痛的发生和发展密切相关。
组蛋白修饰是表观遗传学的重要机制之一,包括乙酰化、甲基化、磷酸化以及miRNA的表达等。大多数组蛋白在赖氨酸残基(Lys)中的乙酰化,通常会促进基因转录,而组蛋白的甲基化可以抑制或激活基因转录,取决于发生甲基化的氨基酸残基。例如,组蛋白H3的Lys9或Lys27的甲基化通常与基因抑制有关,而H3的Lys4、Lys36或Lys79的甲基化通常与基因激活有关。SET domain(SETD)家族是一个重要的表观遗传修饰酶蛋白家族,其成员都包含了一个甲基转移酶SET结构域,包括SETD1a,SETD1b,SETD2-9,主要负责组蛋白H3,H4上赖氨酸残基的甲基化水平调控。组蛋白甲基转移酶SET domain containing2(SETD2)是哺乳动物中唯一负责催化基因体上组蛋白H3第36位赖氨酸的三甲基化(H3K36me3)的酶,是组蛋白表观遗传修饰的重要蛋白之一,参与调节多种与染色质相关的生物进程,如DNA复制、转录和修复。
近年来,越来越多的SETD2突变在中枢神经系统疾病患者中被鉴定出来,如自闭症(Autism spectrum disorder,ASD)、过度生长综合征(Overgrowth syndrome),这提示了SETD2在神经发育及其正常生理功能的重要作用。已有报道患者中,具有SETD2新变体的个体常呈现出神经系统发育相关的表型障碍,包括语言和运动延迟、智力障碍、巨头畸形、ASD和过度生长等。在SETD2突变患者中,几个保守序列中的突变使得患者对于疼痛的感受出现了变化,变得敏感或迟钝(图1-2)。在Setd2自发移码突变(p.V2259Cfs*50)患者中,突变导致了终止密码子提前出现,Setd2转录不完全,患者出现较高的痛觉阈值。在另一项研究及患者随访诊断中发现Setd2错义突变(p.P1062L)患者在出现自闭症表型的同时对疼痛反应迟钝,Setd2内含子突变(c.4715+1G>A)患者对疼痛敏感。因此,申请人推测SETD2能够参与疼痛信号的传导,并在神经病理性疼痛发生发展中起着重要作用。
在过去的20年里,几乎没有新的止痛药被FDA批准用于神经性疼痛的治疗。有几个新兴的治疗手段表现出了治疗神经病理性疼痛的潜力。这些靶点包括抑制谷氨酰胺能神经传递,NMDAR拮抗剂,血管紧张素受体2型拮抗剂,以及突触前调节大麻素和人源化抗神经生长因子单克隆抗体。临床治疗中依旧存在镇痛药物选择匮乏的问题,镇痛药物开发市场前景广泛,开发新型镇痛药物或制剂具有广大的医学应用价值和经济价值。本专利针对SETD2功能缺失的患者进行疼痛症状疾病的诊断以及药物治疗开发,为临床治疗疼痛提供新策略。
发明内容
本发明的目的是为克服上述现有技术的不足,提供组蛋白甲基转移酶SETD2作为神经病理性疼痛药物靶点的应用。
为实现上述目的,本发明采用下述技术方案:
1、组蛋白甲基转移酶SETD2作为神经病理性疼痛药物靶点的应用。
野生型SETD2的氨基酸序列如SEQ ID NO.1所示。
SEQ ID NO.1:
MKQLPSQPPPKMGDFYDPEHPTPEEEENEAKIENVQKTGFIKGPIFKGVASSRFLPKGTKT
KVNLEEQGRQKVSFSFSLTKKTLQNRFFTALSNEKQSDSPHSPATPQVDSNPKAKMEAGD
TFPATEESSPPKSRVELGRIHFKKHLLHVTSRPQLATTTTVASLLPPTTQLPVAIAESTVDSPP
SSPPPPPPPPQVSSPSPPAPISEPVALPHPPVTALMTSTPGPLPVDAAVRAQKEPPMKSVPES
LELDMKQDIVSNSLEEHTNQTLKEQADNALQKEDSHIGKEEEVSDGSKISLSSKKASSKK
KSSQFEGTFLGSESDEDSVRTSSSQRSHDLKSSTSIEKERDFKKSSAPSKSEDLGKSSRSKT
ERDDKYFSYSKLERDTRYVSTRCRSERDRRRSRSRSRSDRVSRTSLSYSRSERSHYYDSER
RYHRSSPYRERTRYSRPYTDNRARESSDSEDEYKKTYPRRTSAHSYRDLRTSSSYSKFDRD
CKTETSYLEMERRGKYSSKLERESKRTSEHEAIKRCCSPPNELGFRRGSSYSKHDNNSTSR
YKSALSKSISKSDKFKNSFCCTELNEENKQSHSFSLQTPCSKGSELRTINKIPEREKTGSPSP
SNQLNDSPTFKKLDESPILKPEFIGHDSHESIKELELPKMKNDQLRNFCSIELNVNGSPETE
TDVATFRTSKPDAISMTSDDSVTGSEVSPLIKGCVLSSNGFQSVGRCRERDADDACRQHN
KSKSPFRETEPLLSPHHDKVMSLPVKTIDYSKTLIKEPVDKRHSCCKTKDSDRYCSPNESS
EAENREISSNCFVNVHLDSKTVVCDNRELTDQHSEFTCDGYKQSIGSTSSASLNHFDDLYE
SVGSSGISSLQSLPSGIRCEENTSPALDTVQSKKGIDFLKYVGKETDVVSALPDSGKAFSS
WENRHNMLSGQSLQESQEEGSSTLHERRGRSEVSLDEEEQRGHTHISDDSEVVFPYDLNL
TMEDSDGVTYTLKCDSSGNAPEIVSTVHGDYSASSASSSDESDSEDTESDDSSIPRNRLQS
VVVVPKNSTLPMEETSPCSSRSSQSYRHYSDHWEDGLESRRHAYEEKFESMASKGCSQTE
KFFLHKGTERNPETSYSQFSRKIDNHLPEIAHSQSDGVDSTSHTDVKSDSVGHLNSEDAIR
AKMSRPQELPVYCDDFEDLPNTSRQQMIFSNRPDSSRLGKAELSFSSSCDISRMDGLHSSE
ELRNLGWDFSQQERPTTTYQQPDSSYGTCGTHKYQQSAEHYGGTHDYWQGNGYWDPR
SAGRPPGTGVVYDRIQGQVPDSLTDDREEEENWDQRSGSHFSSPSNKFFFHQKDKGSVQ
APEISSNSIKDSLVINERKDFSKNFEKNDIKERGPPKKRRQELESDSESDGELQARKKVRVE
IEQGQSSVPQHSELVGPSCAMDDFRDPQRWKEFAKLGKMPCYFDLIEENVYLTERKKNKS
HRDIKRMQCECTPLSKDERAQGEVACGEDCLNRLLMIECSSRCPNGDYCSNRRFQRKQH
ADVEVILTEKKGWGLRAAKDLPSNTFVLEYCGEVLDHKEFKARVKEYARNKNIHYYFM
ALKNDEIIDATQKGNCSRFMNHSCEPNCETQKWTVNGQLRVGFFTTKLVPSGSELTFDYQ
FQRYGKEAQKCFCGSANCRGYLGGENRVSIRAAGGKMKKERSRKKDSVDGELEALMEN
GEGLSDKNQVLSLSRLMVRIETLEQKLTCLKLIQNTHSQSCLKSFLERHGLSLLWIWMAE
LGDGRESNQKLQEEIIKTLEHLPIPTKNMLEESKVLPIIQRWSQTKTAVPQLSEGDGYSSEN
TSRAHTPLNTPDPSAKPSTEVDTDTPKKLIFRRLKIISENSMDSAVSDVTSELECKDGKEDL
DQLETVTVEEDEELQSQQLLPSQLCESKVESESTIDVSKLPISEPEADTETEPKDSNGTKLE
ETIAEETPSQDEEEGVSDVESERSQEPPDKTVDISDLATKLLDSWKDLKEVYRIPKKSQTE
KESTVTERGRDTAAFRDQTAPKTPNRSREREPDKQSQNKEKRKRRGSLSPPSSAYERGTK
RPDDRYDTPTSKKKVRIKDRNKLSTEERRKLFEQEVAQREAQKQQQQMQNLGMTSPLPF
DSLGYNASHHPFAGYPPGYPMQAYVDPSNPNAGKVLLPTPSMDPVCSPAPYDHAQPLVG
HSTESLAAPPSVPVVPHGAASVEVSSSQYAAQSESVVHQDSSVPGMPVQTPGPVQGQNY
SVWDSNQQSVSVQPQYSPAQSQATIYYQGQTCSTVYGVTSPYSQTTPPIVQSYAQPSLQYI
QGQQIFTAHPQGVVVQPAAAVTTIVAPGQPQPLQPPEMVVTNNLLDLPPPSPPKPKTIVLPP
NWKTARDPEGKIYYYHVITRQTQWDPPTWESPGDDASLEHEAEMDLGTPTYDENPMKT
SKKPKTAEADTSSELAKKSKEVFRKEMSQFIVQCLNPYRKPDCKVGRITTTEDFKHLARK
LTHGVMNKELKYCKNPEDLECNENVKHKTKEYIKKYMQKFGAVYKPKEDTEL
2、组蛋白甲基转移酶SETD2在制备神经病理性疼痛诊断和治疗药物中的应用。
3、一种神经病理性疼痛诊断和治疗药物,其有效成分为组蛋白甲基转移酶SETD2活化剂。
优选的,所述的组蛋白甲基转移酶SETD2活化剂为NMDA受体抑制剂或拮抗剂。
4、一种大鼠疼痛模型的构建方法,具体方法如下:首先构建包含大鼠setd2特异性shRNA的慢病毒,其shRNA序列为5’-GCCTGAATTTATAGGACATGA-3’,如SEQ ID NO.2所示,阴性对照序列为5’-TTCTCCGAACGTGTCACGT-3’,如SEQ ID NO.3所示;以上序列的多核苷酸导入慢病毒载体LV3(吉玛基因公司,苏州)从而构建相应的慢病毒;大鼠在麻醉状态下进行鞘内置管手术,总长约9cm的PE-10导管从大鼠腰椎L5与L6间隙插入,进深约4~5cm;通过微量注射器对大鼠鞘内注射包含setd2特异性shRNA的慢病毒20μL,1×108TU/mL,敲低正常大鼠DRG和脊髓中SETD2的表达。
5、一种大鼠疼痛模型,是通过前述构建方法得到的。
6、一种利用基因工程小鼠构建疼痛模型的方法,具体方法如下:Setd2fl/fl小鼠(由浙江医科大学曹雪涛院士赠送)与NaV1.8神经元特异性Cre品系SNSCre/+小鼠(由河北医科大学杜肖娜教授赠送)杂交,选择性地敲除DRG神经元中的Setd2,即Setd2条件KO(Setd2-cKO)小鼠;Setd2-cKO小鼠通过其鼠尾DNA进行PCR鉴定,正向引物:
5’-CCTTGTTTGATTTGATCTTTGGT-3’,如SEQ ID NO.4所示,反向引物:5’-TTAACTTTTGTCTTCGTGCCTTT-3’,如SEQ ID NO.5所示;PCR程序为95℃2min,40个循环的95℃15s,56-60℃30s,72℃50s,最后72℃5min。突变条带为618bp,野生型条带为556bp。
7、一种小鼠疼痛模型,是通过前述构建方法得到的。
本发明的有益效果:
本发明公开了SETD2通过调节NMDA受体表达从而影响外周神经元兴奋性,改变了机体对疼痛信号的敏感性,因此可能成为诊断以及治疗疼痛症状疾病的靶点。本发明提供了新的疼痛症状疾病诊断及治疗的靶点,阐明了病理性疼痛的新机制,使用的药物较目前临床在用的阿片类药物、非甾体类抗炎药、抗抑郁药物等,药物依赖与药物不良反应更低,整体安全性更高,针对性更强。具体如下:
(1)组蛋白甲基化是表观遗传学重要机制之一,参与调控多种生物进程并与多种疾病相关。本发明首次创新性地从SETD2-H3K36me3-NMDA受体-体外细胞模型-病理性疼痛动物模型系统研究SETD2影响NMDA受体调节神经病理性疼痛实际应用及理论价值。
(2)本发明率先从分子水平上研究SETD2调控NMDA受体的体内和体外相互作用及其对神经病理性疼痛的影响,揭示SETD2对NMDA受体膜转运及功能的影响以及调节NMDA受体功能的方式和作用机制,阐明SETD2调控NMDA受体表达的信号通路以及相互作用,为SETD2-H3K36me3-NMDA受体信号轴在神经生物学、分子药理学等方面的应用和药物先导分子的设计开发提供理论基础。
(3)组蛋白赖氨酸残基甲基化的动态平衡由组蛋白甲基转移酶和组蛋白去甲基化酶共同调节,参与基因转录调控,在疼痛症状疾病的发生发展中起着重要作用。
根据以往报道,在神经损伤诱导的神经病理性疼痛模型中,H3K9me2(组蛋白甲基转移酶G9a催化的组蛋白甲基化标记)在1型大麻素受体(CB1R)启动子区域的富集升高,并且显著降低了大鼠DRG中CB1R表达水平。使用G9a抑制剂或者敲除Ezmt2基因均能逆转神经损伤引起的CB1R表达降低,提高CB1R拮抗剂的镇痛作用。神经损伤能够增加DRG中Kcna4、Kcnd2、Kcnq2和Kcnma1启动子区域H3K9me2的甲基化水平,提高了G9a的活性,抑制或者选择性敲除Ezmt2能够阻断K离子通道的沉默,阻止慢性疼痛的发展。在神经损伤下,脊髓和DRG中组蛋白甲基转移酶SUV39H1表达上调,并伴随着MOR的表达下降。使用siRNA敲低SUV39H1的mRNA水平或者使用毛壳素(chaetocin)能够减轻SNL导致的痛觉过敏并恢复MOR表达水平。组蛋白甲基转移酶EZH2在疼痛状态下上调,提高了H3K27me3的甲基化水平,能够促进损伤神经、脊髓和前扣带皮层小胶质细胞中促炎介质的转录,并且下调了SOSC3、NrF2等基因的表达,导致神经炎症的发生。EZH2抑制剂如DZNep或者GSK-126能够缓解神经炎症,减轻神经病理性疼痛。组蛋白甲基转移酶SETD7在慢性神经损伤模型中表达上调,催化H3K4me1的甲基化,促进了小胶质细胞中炎症因子如Ccl2、Il-6和Il-1β的表达,敲低SETD7或者使用SETD7特异性抑制剂PFI-2能够抑制神经损伤下炎症因子的增加,从而缓解疼痛。组蛋白甲基转移酶SET7/9在小鼠癌痛模型脊髓中表达上调,鞘内注射赛庚啶(cyproheptadine)能够下调SET7/9的表达,伴随着RANTES表达降低。SNL模型能够提高背角神经元中甲基转移酶MLL1(KMT2A)和WDR5的表达,提高了MLL1-WDR5复合体与H3K4me3在mGluR5基因启动子上的富集。WDR5-0103能够抑制MLL1与WDR5的相互作用,降低了SNL导致的疼痛超敏和mGluR5的表达增加。
故在已有研究中,疼痛状态下组蛋白甲基化水平改变是由组蛋白甲基转移酶的表达水平或者活性改变引起的,并且绝大多数组蛋白甲基转移酶的表达水平在疼痛状态下上调。与已报道的组蛋白甲基转移酶不同的是,组蛋白甲基转移酶SETD2的表达水平在疼痛状态下下调。而且,组蛋白甲基转移酶SETD2敲低或条件性敲除都会引起疼痛发生,运用NMDA受体抑制剂美金刚(临床药物)可有效缓解疼痛。
附图说明
图1是神经病理性疼痛模型大鼠行为学检测。CCI模型大鼠(A)触觉纤维痛、(B)机械痛和(C)热痛阈值变化。CCI模型大鼠脊髓(D)和DRG(E)中SETD家族基因转录水平变化。CCI模型大鼠(F)脊髓和(I)DRG中Setd2的mRNA水平变化。CCI模型大鼠(G)脊髓和(J)DRG中SETD2的蛋白水平变化,(H)、(K)为对应的数据分析图。数据以平均值±SEM表示,*p<0.05,**p<0.01,***p<0.001。
图2是大鼠脊髓、DRG切片中SETD2与NEUN、IB4、GFAP、IBA-1的共定位情况。大鼠(A)脊髓与(C)DRG中SETD2(绿色)与神经元标记物NeuN、感觉神经元标记物IB4、星形胶质细胞标记物GFAP和小胶质细胞标记物IBA-1(红色)以及细胞核标记DAPI(蓝色)的共定位情况。(B)、(D)为对应的共标阳性细胞比例。数据以平均值±SEM表示,*p<0.05,**p<0.01,***p<0.001。
图3是鞘内注射shRNA特异性敲低Setd2引起大鼠疼痛行为。(A)为实验流程图。(B)为鞘内注射慢病毒与生理盐水(阴性对照)对大鼠脊髓与DRG中细胞的感染情况。鞘内注射shRNA特异性敲低Setd2对大鼠(C)触觉纤维痛、(D)机械痛和(E)热痛阈的影响。荧光定量PCR检测大鼠(F)脊髓与(I)DRG中基因的转录水平。Western blot检测大鼠(G)脊髓与(J)DRG中目的蛋白的表达水平,(H)、(K)为对应的数据分析图。数据以平均值±SEM表示,*p<0.05,**p<0.01,***p<0.001。
图4是抑制NMDA受体缓解大鼠的痛性行为。(A)为实验流程图。腹腔注射MEM对大鼠(B)机械纤维痛、(C)压力痛、(D)热痛觉阈值的影响。鞘内注射AP-5对大鼠(E)机械纤维痛、(F)压力痛、(G)热痛觉阈值的影响。数据以平均值±SEM表示,*p<0.05,**p<0.01,***p<0.001。
图5是通过Cre-loxp重组酶系统条件性敲除小鼠DRG中的SETD2并检测其基础痛阈。(A)为小鼠构建流程图。通过(B)免疫荧光和(C)荧光定量PCR检测小鼠脊髓与DRG中SETD2的表达水平。(E)棉签实验检测小鼠的非伤害性机械刺激感受。(F)、(G)毛刷实验检测小鼠的动态伤害性机械刺激感受。(H)、(I)针刺实验检测小鼠的伤害性机械刺激感受。(J)、(K)热板检测小鼠在不同温度下的热痛觉感受。(L)、(M)冷板检测小鼠在不同温度下的冷痛觉感受。数据以平均值±SEM表示,*p<0.05,**p<0.01,***p<0.001。
图6是抑制NMDA受体缓解小鼠的痛性行为。小鼠的(A)机械纤维痛阈和(B)热痛阈。腹腔注射组蛋白去甲基化酶抑制剂JIB-04对小鼠的(C)机械纤维痛阈和(D)热痛阈的影响。通过荧光定量PCR检测小鼠(E)脊髓与(H)DRG中目的基因的转录水平变化。通过Westernblot检测小鼠(F)脊髓与(I)DRG中目的蛋白的表达水平变化,(G)、(J)为对应的数据分析图。腹腔注射MEM对小鼠的(K)机械纤维痛阈和(L)热痛阈的影响。数据以平均值±SEM表示,*p<0.05,**p<0.01,***p<0.001。
具体实施方式
下面结合附图和实施例对本发明进行进一步的阐述,应该说明的是,下述说明仅是为了解释本发明,并不对其内容进行限定。
实验动物:
SPF级斯普拉格-道利(SD)雄性大鼠,体重250-300g购于湖南斯莱克景达实验动物公司,饲养于中南大学实验动物中心屏障环境内,保持室温(24±1℃)以及12小时昼/夜循环,自由饮水和进食。
SPF级Setd2-loxp和SNS-cre小鼠遗传背景为C57/BL/6,6-8周周龄,饲养于中南大学实验动物中心屏障环境内,保持室温(24±1℃)以及12小时昼/夜循环,自由饮水和进食。所有实验操作均遵循国际疼痛研究协会伦理委员会的规定。
实验方法:
1、荧光定量PCR技术以及Western blot技术进行表达水平检测。动物组织RNA提取采用RNA纯化试剂盒(#EZB-RN001,),RNA逆转录成cDNA使用HiScriptⅡQ RTSuperMix for qPCR(#R222-01,南京诺维赞生物公司),RT-qPCR使用Universal SYBRqPCRMaster Mix(#Q712-02,南京诺维赞生物公司),所用引物如表1,所有操作均按照说明书进行。动物组织蛋白提取使用RIPA裂解液(碧云天生物有限公司),提取的蛋白通过BCA试剂盒进行定量(#P0012,碧云天生物有限公司),所有操作均按照说明书进行。每个孔道40μg蛋白上样量使用SDS-PAGE凝胶电泳进行分离并转至PVDF膜上。PVDF膜通过5%脱脂牛奶室温封闭1h并在4℃与相应的一抗孵育过夜。所用一抗及对应稀释比例为SETD2(1:1000,#BS7519,Bioworld),β-actin(1:10000,#ABM40032,Abbkine),GAPDH(1:10000,#60004-1-lg,Proteintech),GluN1(1:1000,#bs-23343R,Bioss),H3K36me3(1:1000,#A2366,Abcolonal),histone H3(1:10000,#17168-1-AP,Proteintech)。条带洗涤3次后与对应的二抗室温孵育1h。所用二抗及对应稀释比例为HRP conjugated goat anti-rabbit IgG(1:20000,#511203,Zenbio),goat anti-mouse IgG(1:20000,#511103,Zenbio)。最后使用ECL化学发光底物(#BL520A,Biosharp)在化学发光仪中进行发光。
表1荧光定量PCR引物序列
2、组织切片、免疫荧光及激光共聚焦技术观察目的蛋白质分布和定位。动物在麻醉状态下经心脏灌注生理盐水和质量浓度4%多聚甲醛。取出其脊髓L4-6腰段及对应的DRG,质量浓度4%多聚甲醛固定24h,20-30%(w/v)蔗糖溶液梯度脱水。脱水后的组织使用OCT包埋剂(SAKURA)包埋,在冰冻切片机(#FS800A,RWD)中切成10μm厚度的组织切片。组织切片使用0.2%的PBST(#T8200,Solarbio)洗涤3次,用3%BSA(m/v,#9048-46-8,Beyotime)和10%山羊血清(v/v,#AR0009,BOSTER)室温封闭1h。使用下列一抗4℃孵育过夜:rabbitanti-SETD2(1:200,#BS7519,Bioworld),mouse anti-NeuN(1:200,#3A4C1,Proteintech),mouse anti-GFAP(1:200,#3670,CST),mouse anti-IBA1(1:200,#MA5-27726,Invitrogen),mouse anti-FLAG(1:200,#K200001M,Solarbio),rabbit anti-IgG(1:200,#A7508,Beyotime),Dylight 594labeled GSLⅠ-isolectin B4(IB4,1:200,#MP6316,Maokang Biotechnology)。然后用PBST洗涤3次,室温孵育荧光标记二抗1h:goat anti-rabbit IgG(H+L)conjugated withAlexaFluor 488(1:200,#A-11008,Invitrogen),goatanti-mouse IgG(H+L)conjugated withAlexaFluor 568(1:200,#A-11004,Invitrogen)。最后使用抗荧光淬灭剂进行封片。
3、大鼠CCI神经病理性疼痛模型构建:选用雄性SD大鼠,质量200-220g。随机均分为2组:慢性坐骨神经压迫损伤(Chronic Constriction Injury,CCI)组、假手术组(Sham组,不结扎压迫坐骨神经,其他手术步骤同CCI组)。大鼠在4%异氟烷麻醉下,钝性分离其肌肉,暴露其左侧坐骨神经,使用4-0不可吸收手术缝线在其坐骨神经上轻轻地打4个结,间隔1mm左右,再将神经塞回原处,缝皮。
4、疼痛行为检测:(1)为了检测触觉异常疼痛,申请人将电子Von-frey仪(IITC)应用于动物的后爪中心部位。实验动物单独放置在网地板上的单独室中适应30min后,将Von-frey探头垂直于后爪的足底中心,施加力并观察动物出现抬脚反应。通过读取表上读数并计算最终触觉刺激产生50%的抬腿反应的阈值。为了量化实验动物的机械伤害感受阈,申请人用压力测痛仪对动物后爪进行了爪压试验。随着施加力的增加,当动物发出声音或抽出爪子而表现出疼痛时,立即释放踏板,并在量表上读取此时动物的压痛阈值。(2)为了测量动物的热痛阈,将动物放在热测试仪的玻璃表面的单独室中,待其适应30min后,移动位于玻璃下方的移动辐射热源(Ugo Basile)集中在动物的后爪中心。在恒定温度刺激下记录后爪的抬爪潜伏期,并对每只后爪进行两次测试,得到平均值。
5、Setd2特异性shRNA慢病毒鞘内注射。在异氟烷诱导麻醉过程中,申请人首先在大鼠体内植入鞘内导管。简单地说,麻醉后的动物被放置在立体定位架上,在其腰椎L5处做了一个小切口,用腰麻针引导穿刺插入一根长度约5cm的PE-10导管,微量注射器进行注射。大鼠Setd2特异性shRNA慢病毒或阴性对照慢病毒(序列如上所述),最终滴度为10^8TU/mL,用于鞘内注射(20μL)。在慢病毒注射后7天至28天持续观察阴性对照病毒组与Setd2特异性敲低组动物疼痛行为的变化。
6、Setd2条件性敲除小鼠的建立及鉴定。Setd2fl/fl小鼠(由浙江医科大学曹雪涛院士赠送)与NaV1.8神经元特异性Cre品系SNSCre/+小鼠(由河北医科大学杜肖娜教授赠送)杂交,选择性地敲除DRG神经元中的Setd2,即Setd2条件KO(Setd2-cKO)小鼠。Setd2-cKO小鼠通过其鼠尾DNA进行PCR鉴定,正向引物:5’-CCTTGTTTGATTTGATCTTTGGT-3’,如SEQ ID NO.4所示,反向引物:5’-TTAACTTTTGTCTTCGTGCCTTT-3’,如SEQ ID NO.5所示;PCR程序为95℃2min,40个循环的95℃15s,56-60℃30s,72℃50s,最后72℃5min。突变条带为618bp,野生型条带为556bp。在基因型鉴定为cKO小鼠后,还需进行以下实验:a、Western blot检测cKO小鼠DRG神经元目的蛋白表达减少情况;b、通过免疫荧光双标技术观察神经元中目的蛋白的敲除情况;c、采用转棒实验(Rotarod)观察cKO小鼠有无运动功能异常。分别检测Setd2fl/fl型与Setd2条件敲除小鼠疼痛行为,观察敲除Setd2对动物痛性行为的影响。
实验结果:
1、慢性神经病理性疼痛模型中目的基因的表达变化
图1中A-C是大鼠坐骨神经慢性压迫损伤模型(CCI)行为学测试。通过隔天对大鼠的痛觉阈值检测结果发现,CCI模型大鼠在模型构建后第3天开始,机械缩爪阈值(PawWithdrawal Threshold,PWT)显著降低,压力痛觉阈值(Pressure)显著降低,缩爪潜伏期(Paw Withdrawal Latency,PWL)显著降低,大鼠痛性行为明显,第10天左右模型逐渐稳定(图1中A-C,p<0.05)。
图1中D-E是在大鼠CCI模型构建第14天,对其脊髓以及背根神经节(Dorsal RootGanglion,DRG)中SETD家族基因转录水平变化进行检测。结果显示,setd1b,setd3和setd5在脊髓中的转录水平明显上升,setd2在脊髓和DRG中的转录水平明显下降,setd7在脊髓和DRG中的转录水平明显上升(图1中D、E,p<0.05),提示SETD2在神经病理性疼痛中的重要作用以及其作为疼痛症状诊断及治疗的靶点潜力。
图1中F-K是为了确定神经损伤是如何影响SETD2在大鼠外周神经系统中变化的,对CCI模型大鼠脊髓L4-L6腰段以及对应的DRG进行了SETD2表达水平检测。荧光定量PCR结果表明,术后3、5、7和14天,CCI大鼠脊髓与DRG中SETD2的mRNA水平显著低于假手术对照组(图1中F、I,p<0.05)。Westernblot结果显示,术后7、14天,CCI大鼠脊髓中SETD2的蛋白水平显著低于假手术对照组(图1中G、H,p<0.05);术后5、7和14天,CCI大鼠DRG中SETD2的蛋白水平显著低于假手术对照组(图1中J、K,p<0.05)。CCI模型中,大鼠外周神经系统中SETD2的转录与蛋白水平与其痛性行为呈负相关,进一步说明SETD2在神经病理性疼痛发生、发展过程中持续发挥作用,是疼痛信号传导过程中组蛋白修饰改变的重要蛋白之一。
2、目的基因与外周神经系统中神经元共定位
图2是通过免疫荧光标记法测定SETD2在大鼠脊髓与DRG中的分布。免疫荧光结果表明,用SETD2抗体进行免疫染色,申请人发现大鼠脊髓与DRG神经元中有天然的SETD2蛋白(绿色)表达,并与神经元标记物NeuN(红色)共定位(图2中A、C),这种共定位在放大的神经元图像上尤为明显。在大鼠脊髓切片中,SETD2的免疫反应(绿色)在大鼠脊髓背角中有大量分布,并与神经元中的NeuN(红色)共定位。免疫荧光结果显示,SETD2分布于脊髓中不同大小的神经元中,在脊髓Ⅰ、Ⅱ层(绿色)与IB4标记的终末(红色)共定位。免疫荧光结果显示,SETD2存在于大鼠不同大小的DRG神经元中,小直径神经元中SETD2(绿色)与IB4受体(红色)也存在免疫共定位。SETD2与小胶质细胞与星形胶质细胞的标记物IBA1和GFAP共定位较少,主要与神经与共定位(图2中B、D)。提示SETD2主要在大鼠外周神经系统中发挥作用,通过调控神经元兴奋性参与疼痛信号传导。
3、敲低目的基因引起大鼠痛觉过敏
图3是为了确定SETD2在痛觉控制中的作用,采用鞘内注射包含setd2特异性shRNA的慢病毒,敲低正常大鼠DRG和脊髓中SETD2的表达,并对慢病毒处理的大鼠进行疼痛行为学实验。实验示意图如图3中A所示。利用免疫荧光对慢病毒感染细胞进行标记,结果显示,慢病毒注射组较生理盐水对照组荧光强度明显增强,且IgG无明显变化,表明慢病毒能够感染脊髓与DRG中大部分细胞(图3中B)。
从慢病毒注射后的一周开始,隔天测量它们对机械刺激、伤害性压力和热辐射的反应阈值。鞘内注射setd2特异性shRNA慢病毒的大鼠,相较于阴性对照慢病毒注射组,大鼠的PWT、Pressure和PWL逐渐降低,表明存在着机械、压力和热痛觉过敏(图3中C-E,p<0.05)。
为了验证setd2特异性shRNA的慢病毒的作用,申请人在慢病毒注射28天后对大鼠腰段L4-L6脊髓和DRG中差异基因的mRNA水平进行了检测。荧光定量PCR结果显示,鞘内注射setd2敲低病毒后,脊髓中setd2基因转录水平显著下调;编码NMDA受体的grin1、grin2a和grin2b基因转录水平上调(图3中F,p<0.05)。DRG中setd2基因转录水平显著下调;编码NMDA受体的grin1基因转录水平上调(图3中I,p<0.05)。Western blot结果显示注射敲低病毒后,脊髓与DRG中SETD2蛋白水平显著下调,H3K36me3甲基化水平显著下降,GluN蛋白水平显著上升(图3中G、J)。这些结果提示敲低setd2可能通过影响其组蛋白甲基转移酶的活性导致了NMDA受体的过度转录激活,引发大鼠痛觉过敏。数据分析图见图3中H、K。
4、抑制NMDA受体缓解大鼠痛性行为
图4是为了确证NMDA受体在敲低setd2引起的大鼠痛觉过敏中的作用,实验流程图如图4中A所示,申请人在慢病毒注射大鼠中腹腔注射NMDA受体拮抗剂Memantine(MEM),在0-2h的作用时间内,较溶剂组相比,MEM可以显著减轻setd2特异性敲低慢病毒引起的大鼠痛觉阈值降低(图4中B-D,p<0.05);鞘内注射NMDA受体拮抗剂AP5,在0-2h的作用时间内,较溶剂组相比,AP5可以显著减轻setd2特异性敲低慢病毒引起的大鼠痛觉阈值降低(图4中E-G,p<0.05)。行为学结果显示,抑制脊髓中NMDA受体的活性,能够有效减轻敲低setd2引起的大鼠痛觉过敏,提示SETD2在神经病理性疼痛中的作用机制NMDA受体密切相关。
5、条件敲除目的基因小鼠出现痛觉过敏
图5是利用Setd2-floxp小鼠和SNS-cre小鼠来条件性敲除小鼠DRG中Setd2的表达(图5中A)。免疫荧光结果显示条件敲除小鼠的DRG中SETD2水平显著降低,脊髓背角中SETD2水平显著降低(图5中B)。荧光定量PCR结果显示,敲除Setd2后,脊髓和DRG中Setd2基因转录水平显著下调(图5中C、D)。
行为学检测结果显示,非伤害性感受刺激阈值没有明显改变(图5中E),机械性伤害感受阈明显降低(图5中F-I),热痛觉与冷痛觉阈值显著降低(图5中J-M),小鼠表现出痛觉过敏现象。
6、抑制NMDA受体缓解条件敲除小鼠痛性行为
图6是对条件敲除小鼠的行为学检测结果,显示小鼠出现明显的机械痛和热痛敏(图6中A、B)。申请人对小鼠腹腔注射组蛋白去甲基化酶抑制剂JIB-04,在0-2h的作用时间内,较溶剂组相比,可以减轻敲除Setd2引起的痛觉阈值降低(图6中C、D)。这表明敲除Setd2引起痛觉过敏可能是通过影响其组蛋白甲基转移酶活性导致的。
为了检测Setd2特异性敲除后小鼠DRG和脊髓中NMDA受体表达水平的变化,进行了荧光定量PCR结果显示,敲除Setd2后,DRG中grin1基因转录水平显著上调(图6中E,p<0.05);脊髓中grin1基因转录水平显著上调(图6中M,p<0.05)。Westernblot结果显示敲除Setd2后,脊髓与DRG中SETD2蛋白水平显著下调,H3K36me3甲基化水平显著下降,GluN蛋白水平显著上升(图6中F、G)。这些结果提示敲除Setd2可能通过影响其组蛋白甲基转移酶的活性导致了NMDA受体的过度转录激活,引发小鼠痛觉过敏。
为了确证NMDA受体在Setd2条件敲除鼠痛觉过敏中的作用,腹腔注射NMDA受体拮抗剂MEM,在0-2h的作用时间内,较溶剂组相比,MEM可以显著减轻Setd2特异性敲除引起的小鼠痛觉阈值降低(图6中K、L)。行为学结果显示,抑制脊髓中NMDA受体的活性,能够有效减轻敲除Setd2引起的大鼠痛觉过敏,提示SETD2在神经病理性疼痛中的作用机制与NMDA受体密切相关。
上述虽然结合附图对本发明的具体实施方式进行了描述,但并非对本发明保护范围的限制,在本发明的技术方案的基础上,本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。
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Claims (7)
1.组蛋白甲基转移酶SETD2作为神经病理性疼痛药物靶点的应用。
2.组蛋白甲基转移酶SETD2在制备神经病理性疼痛诊断和治疗药物中的应用。
3.一种神经病理性疼痛诊断和治疗药物,其特征在于,其有效成分为组蛋白甲基转移酶SETD2活化剂。
4.一种大鼠疼痛模型的构建方法,其特征在于,具体方法如下:首先构建包含大鼠setd2特异性shRNA的慢病毒,其shRNA序列为5’-GCCTGAATTTATAGGACATGA-3’,如SEQ IDNO.2所示,阴性对照序列为5’-TTCTCCGAACGTGTCACGT-3’,如SEQ ID NO.3所示;以上序列的多核苷酸导入慢病毒载体LV3从而构建相应的慢病毒;大鼠在麻醉状态下进行鞘内置管手术,总长约9cm的PE-10导管从大鼠腰椎L5与L6间隙插入,进深约4~5cm;通过微量注射器对大鼠鞘内注射包含setd2特异性shRNA的慢病毒20μL,1×108TU/mL,敲低正常大鼠DRG和脊髓中SETD2的表达。
5.一种大鼠疼痛模型,其特征在于,是通过权利要求5所述构建方法得到的。
6.一种利用基因工程小鼠构建疼痛模型的方法,其特征在于,具体方法如下:Setd2fl/fl小鼠与NaV1.8神经元特异性Cre品系SNSCre/+小鼠杂交,选择性地敲除DRG神经元中的Setd2,即Setd2-cKO小鼠;Setd2-cKO小鼠通过其鼠尾DNA进行PCR鉴定,正向引物:5’-CCTTGTTTGATTTGATCTTTGGT-3’,如SEQ ID NO.4所示,反向引物:5’-TTAACTTTTGTCTTCGTGCCTTT-3’,如SEQ ID NO.5所示;PCR程序为95℃2min,40个循环的95℃15s,56-60℃30s,72℃50s,最后72℃5min。
7.一种小鼠疼痛模型,其特征在于,是通过权利要求6所述构建方法得到的。
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