CN110585228B - LncRNA PTPRE-AS1在制备抑制蟑螂提取物诱导的过敏性哮喘药物中的应用 - Google Patents
LncRNA PTPRE-AS1在制备抑制蟑螂提取物诱导的过敏性哮喘药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种LncRNA PTPRE‑AS1在制备抑制蟑螂提取物诱导的过敏性哮喘药物中的用途。在小鼠骨髓来源的原代巨噬细胞中，PTPRE‑AS1可以通过显著降低ERK1/2的磷酸化进而抑制IL‑4诱导的下游M2型巨噬细胞基因表达，在IL‑4诱导的M2型巨噬细胞极化中，PTPRE‑AS1 起着负向调控的作用。在蟑螂提取物诱导的小鼠哮喘模型中，PTPRE‑AS1敲除小鼠的肺部炎症明显加重。同时，PTPRE‑AS1的表达在哮喘儿童的PBMCs中显著降低。本发明的PTPRE‑AS1可作为过敏性哮喘干预治疗的一个新靶点。
Description
技术领域
本发明涉及长链非编码RNA领域，具体是一种LncRNA PTPRE-AS1在制备抑制蟑螂提取物诱导的过敏性哮喘药物中的应用。
背景技术
巨噬细胞作为一种初始免疫细胞，是机体炎症反应的重要参与和调节者。巨噬细胞具有明显的异质性和可塑性，在不同的环境条件下可以分化为经典激活(classicallyactivated)的M1型和替代激活(alternatively activated)的 M2型两种功能截然不同的亚型，即巨噬细胞的极化。M1型巨噬细胞主要由 LPS、IFN-γ等激活，可诱导Th1细胞反应，主要参与抗感染免疫应答，并引起急性炎症损伤。M2型巨噬细胞主要由IL-4、IL-13、IL-10等激活，可诱导Th2细胞反应，从而发挥调节性或抑制性效应对抗急性炎症反应,参与组织修复功能。巨噬细胞的极化状态直接关系到多种炎症和免疫相关性疾病的发生、发展和转归：例如难治性哮喘、炎症性肠病、动脉粥样硬化、胰岛素抵抗型糖尿病等。因此探讨如何调控巨噬细胞极化是当今生物医学研究的重点和难点，并可能为上述疾病的治疗提供新策略，具有重要意义。
长链非编码RNA(long non-coding RNA,lncRNA)是一类长度大于200bp 的不具有编码蛋白功能的RNA，可通过染色质重塑、转录水平的调节和转录后水平的调节等一系列的调控机制来调节基因的表达。在免疫系统中， lncRNA广泛地表达在单核细胞﹑巨噬细胞﹑中性粒细胞﹑淋巴细胞等免疫细胞中，并在细胞的发育﹑分化和活化过程中有着重要作用。例如，近来的一项研究揭示lncRNA NeST的转录基因位于干扰素-γ(interferon-gamma,IFNG)的反义链，且两个基因转录方向相反，NeST可以与H3K4甲基化转移酶复合物的成分WDR5结合，调节IFNG位点的组蛋白甲基化，促进IFNG 的表达，控制机体对病原微生物的易感性。
近年来，lncRNA对巨噬细胞极化的调控日益引起人们的关注。但目前的研究多集中于lncRNA对M1型巨噬细胞极化调控的影响，对lncRNA在 M2型巨噬细胞极化中的作用还知之甚少。
发明内容
本发明就是为了提供一种预防、诊断和治疗过敏性哮喘的新途径，所提出一种LncRNA PTPRE-AS1在制备抑制蟑螂提取物诱导的过敏性哮喘药物中的应用。
本发明是按照以下技术方案实现的。
一种LncRNA PTPRE-AS1在制备抑制蟑螂提取物诱导的过敏性哮喘药物中的用途。
进一步的，PTPRE-AS1参与负向调控IL-4诱导的M2型巨噬细胞极化。
进一步的，PTPRE-AS1通过显著降低ERK1/2的磷酸化进而抑制IL-4诱导的下游M2型巨噬细胞基因表达。
进一步的，PTPRE-AS1敲除导致小鼠的过敏性哮喘病情加重。
进一步的，PTPRE-AS1的表达在哮喘儿童的PBMCs中显著降低。
一种治疗过敏性哮喘药物的药物，所述药物的有效成分中包括LncRNA PTPRE-AS1。
本发明获得了如下的有益效果。
本发明通过分离培养小鼠骨髓来源的原代巨噬细胞并由IL-4诱导为M2 型巨噬细胞，采用RNA高通量测序首次筛选出异常高表达的PTPRE-AS1,并发现PTPRE-AS1参与负向调控IL-4诱导的M2型巨噬细胞极化；同时在 PTPRE-AS1敲除小鼠中发现蟑螂提取物诱导的肺部炎症明显加重。本发明将为进行过敏性哮喘的预防、诊断和治疗提供一个新的途径。
附图说明
图1是本发明PTPRE-AS1在IL-4诱导的M2型巨噬细胞中的表达水平图；
图2是本发明PTPRE-AS1对IL-4诱导的M2型巨噬细胞极化的影像图；
图3是本发明PTPRE-AS1敲除对蟑螂提取物诱导的小鼠过敏性哮喘的影响图；
图4是本发明PTPRE-AS1在儿童过敏性哮喘PBMCs中的表达水平图。
具体实施方式
下面结合附图及实施例对本发明进行进一步说明。
一、材料和方法：
1.小鼠骨髓来源的原代巨噬细胞(bone marrow-derived macrophages， BMDMs)的培养和转染
从C57/B6的小鼠股骨中提取骨髓，裂解红细胞，PBS洗涤2次，离心去上清，培养于DMEM(加10％小牛血清，加20％F12,加1.5％HEPES,加100U/ml 青霉素-链霉素，加10ng/mlM-CSF)，培养至7-8天，传代使用。
小鼠BMDMs中使用的PTPRE-AS1过表达(PTPRE-AS1 LV)和敲底病毒 (PTPRE-AS1shRNA)及相应的阴性对照来自上海吉玛公司 (http://www.genepharma.com/operation_ manual2.php？cateid＝151)。
2.RNA抽提、逆转录和实时荧光定量PCR(RT-qPCR)
小鼠BMDMs培养于24孔,各加入5μL滴度为108U/mlPTPRE-AS1 LV和PTPRE-AS1shRNA病毒以及相应对照,72hr后加入IL-4(20ng/ml，R&D公司),刺激24hr后,除去上清，使用酚氯仿法抽提RNA。同时小鼠的肺组织进行组织研磨并且也采用酚氯仿法抽提RNA。
应用酚氯仿法抽提Trizol(Invitrogen公司)得到的RNA用毛细管电泳(NanoDropSpecthophotometer)鉴定其完整性和测定其浓度。mRNA的逆转录采用PrimeScript RTreagent kit(TaKaRa公司),逆转录后的cDNA 经SYBR Premix Ex Taq RT-PCR kit(TaKaRa公司)扩增，并在ABI ViiA7 测序仪(Applied Biosystems公司)上进行实时荧光定量PCR操作。
3.免疫印迹实验(western blotting)
小鼠BMDMs培养于6孔,各加入10μL滴度为108U/mlPTPRE-AS1 LV和 PTPRE-AS1shRNA病毒以及相应对照,72hr后加入IL-4(20ng/ml，R&D公司),刺激24hr后,除去上清，应用RIPA Buffer(Thermo公司)裂解法抽提总蛋白。采用抗体pERK 1/2(CST公司)，ERK 1/2(CST公司)，PTPRE(CST 公司)，β-tubulin(abcam公司)。
4.RNA原位杂交实验(FISH)
采用Stellaris公司的RNA FISH kit，小鼠BMDMs培养于6孔,用固定液固定细胞10mins，以及70％乙醇进行透膜，加入PTPRE-AS1探针 (5’-gtgtgagaggtcgttttttc-3’和5’-agatcatagctcctctggac-3’)和 DAPI孵育，最后在Leica公司激光共聚焦显微镜下观察。
5.蟑螂提取物诱导的小鼠过敏性哮喘模型实验
PTPRE-AS1敲除小鼠以及对照C57BL/6小鼠由上海南方模式公司构建以及提供(https://www.modelorg.com/models-service/genetically-modified-mouse-models- generation/genetic-modification/gene-knockout.html#KO)，通过CRISPR基因编辑技术，敲除PTPRE-AS1的外显子2和3序列从而导致PTPRE-AS1体内功能缺失，所有小鼠均在SPF环境下饲养。
选取6-8周龄的C57BL/6小鼠，分别在第0天、1天、2天、14天将蟑螂提取物(CRE)通过气管注射入小鼠体内，并在第20天和22天再次气管刺激，于第23天收集肺泡灌洗液(Bronchoalveolar lavage fluid,BALF)，通过流式分析检测灌洗液中的炎症细胞数量；安乐死小鼠，收集肺组织进行随后的实验检测。
6.小鼠肺组织病理分析
将小鼠的肺组织用中性甲醛固定，进行石蜡切片，采用HE染色，分析其病理变化。
7.正常和过敏性哮喘儿童PBMCs分离收集
收集正常和过敏性哮喘儿童的全血，与等体积的PBS混合后，采用GE 公司的Ficoll-Paque(p＝1.077g/ml)进行梯度离心，吸取中间的一圈的白色细胞即为PBMCs。
二、结果：
1.PTPRE-AS1在IL-4诱导的M2型巨噬细胞中高表达
使用UCSC和NCBI网站检索发现PTPRE-AS1(Gene Accession number: NR_015548)作为非编码RNA，位于小鼠的7号染色体，在PTPRE基因的反义链上，且两个基因转录方向相反。将小鼠的BMDMs进行3hr LPS或24hr IL-4 刺激后，与对照相比，发现IL-4能够显著提高PTPRE-AS1的表达水平(图 1A)。同时通过RNA FISH实验，发现在小鼠的BMDMs中，PTPRE-AS1在细胞核中高表达(图1B)。
图1.PTPRE-AS1的表达水平在IL-4诱导的M2型巨噬细胞中显著升高。 (A)将小鼠的BMDMs进行LPS 3hr或IL-4 24hr处理，以及不处理作为对照，RT-qPCR检测PTPRE-AS1的表达水平；(B)将小鼠的BMDMs固定及透膜，采用Quasar 670染料探针标记PTPRE-AS1，检测PTPREE-AS1在细胞核和细胞质中的分布。
2.PTPRE-AS1显著抑制IL-4诱导的M2型巨噬细胞极化
在小鼠的BMDMs中，分别采用PTPRE-AS1 shRNA和PTPRE-AS1 LV慢病毒对PTPRE-AS1进行敲低和过表达。与对照相比，发现降低PTPRE-AS1的表达能够显著提高IL-4诱导的IL-10﹑Arg-1和CD206等M2型巨噬细胞极化基因的表达水平，而促进PTPRE-AS1的表达能够显著抑制IL-4诱导的M2型巨噬细胞极化基因的表达水平(图2A和2B)；进一步数据显示在IL-4刺激的BMDMs中，PTPRE-AS1参与正向调控其宿主基因PTPRE的表达(图2C)；
同时，在IL-4诱导的M2型巨噬细胞极化过程中，MAPK/ERK1/2信号通路在其中扮演着重要作用。通过western blotting，与对照相比，发现降低 PTPRE-AS1的表达能够显著促进IL-4诱导的ERK1/2蛋白的磷酸化水平，对 ERK1/2的总蛋白表达无影响；而促进PTPRE-AS1的表达能够显著抑制IL-4 诱导的ERK1/2蛋白的磷酸化水平，对ERK1/2的总蛋白表达也无影响(图2D)。以上结果表明PTPRE-AS1可以通过抑制IL-4诱导的MAPK/ERK1/2信号通路的活化进而负向调控M2型巨噬细胞的极化。
图2.PTPRE-AS1显著抑制IL-4诱导的M2型巨噬细胞极化。(A)(B) (C)在小鼠的BMDMs中分别加入PTPRE-AS1 shRNA和PTPRE-AS1LV慢病毒， 72hr后,IL-4处理24hr，RT-qPCR检测M2型巨噬细胞极化基因和宿主基因 PTPRE的表达水平；(D)在小鼠的BMDMs中分别加入PTPRE-AS1 shRNA和 PTPRE-AS1 LV慢病毒，72hr后,IL-4处理，western blotting检测ERK1/2 蛋白磷酸化和总蛋白的表达水平。
3.在蟑螂提取物诱导的过敏性哮喘小鼠模型中，PTPRE-AS1敲除导致小鼠的肺部组织炎症加重
在蟑螂提取物诱导的过敏性哮喘模型中，收集肺泡灌洗液(BALF)，通过流式分析检测灌洗液中的总细胞和炎症细胞数量，发现经CRE处理后，在 PTPRE-AS1敲除小鼠中总的炎症细胞数量增加，尤其嗜酸性粒细胞和巨噬细胞数目明显增多(图3A)；用4％多聚甲醛固定肺组织，随后石蜡切片，用于H&E染色，检测肺组织的病理状态。与对照组(WT+CRE)相比，发现在 PTPRE-AS1敲除小鼠中，肺部组织有非常明显的组织病理损伤，有着大量炎症细胞的浸润(图3B)；同时发现经CRE处理后,在PTPRE-AS1敲除小鼠中，肺部组织的PTPRE基因表达水平与对照组(WT+CRE)相比明显降低(图3C)。综上，以上实验结果表明PTPRE-AS1敲除可以导致小鼠的过敏性哮喘病情加重。
图3.PTPRE-AS1敲除加重蟑螂提取物诱导的小鼠过敏性哮喘的发生发展。(A)将实验小鼠随机分为4组；WT+PBS处理，PTPRE-AS1敲除+PBS处理，WT+CRE处理，PTPRE-AS1敲除+CRE处理，每组5只小鼠。于第23天，流式检测小鼠肺泡灌洗液中的总细胞和炎症细胞数量；(B)获取小鼠的肺组织，进行HE染色，对其病理损伤进行评估；(C)获取小鼠的肺组织,匀浆进行RNA抽提，RT-qPCR检测PTPRE-AS1和其宿主PTPRE基因的表达水平。
4.PTPRE-AS1的表达水平在过敏性哮喘儿童的PBMCs中显著降低
通过从复旦大学附属儿科医院收集正常儿童和过敏性哮喘儿童的PBMCs 样本,并进行RT-qPCR检测，发现PTPRE-AS1和其宿主基因PTPRE在过敏性哮喘儿童的PBMCs中的表达水平都显著降低(图4A)；同时通过ROC曲线分析，发现PTPRE-AS1和其宿主基因PTPRE与过敏性哮喘儿童的临床特异性密切相关(图4B)。总之，以上结果表明PTPRE-AS1有可能作为儿童过敏性哮喘的生物标志物之一，用于哮喘的预防、诊断和治疗。
图4.PTPRE-AS1表达水平在儿童过敏性哮喘的PBMCs中显著降低。(A) 收集40例正常和42例过敏性哮喘儿童的PBMCs样本，RT-qPCR检测 PTPRE-AS1和PTPRE基因的表达水平；(B)对PTPRE-AS1和PTPRE基因与过敏性哮喘的特异性进行ROC曲线分析。
三、讨论
在小鼠骨髓来源的原代巨噬细胞(BMDMs)中，发现IL-4可以促进长链非编码RNAPTPRE-AS1的表达，而高表达的PTPRE-AS1又可以通过抑制IL-4 诱导的MAPK/ERK1/2的信号通路激活进而负向调控IL-4诱导的M2型巨噬细胞极化。进一步地，在蟑螂提取物诱导的小鼠过敏性哮喘模型中，验证了PTPRE-AS1的敲除可以导致小鼠的肺部炎症加重；同时，在过敏性哮喘儿童的PBMCs中检测到低表达的PTPRE-AS1，以上结果揭示PTPRE-AS1参与到过敏性哮喘的发生发展过程中。
现今的大量研究揭示IL-4诱导的M2型巨噬细胞在阻抗炎症反应、组织修复以及伤口愈合等生理病理活动过程中发挥着重要作用，并且参与到过敏性哮喘等多种疾病的发生发展过程中。目前，研究发现哮喘是一种慢性炎症反应的气道疾病，常伴有粘液分泌增加和气道重塑从而导致出现喘息、咳嗽和胸闷等症状。哮喘的发病机理复杂，主要是由于Th2细胞驱动的炎症反应紊乱，其中IL-4和IL-13作为关键的Th2型细胞因子参与到嗜酸性粒细胞的浸润、IgE抗体产生以及组胺的释放等多项病理活动过程，从而在哮喘的发生发展中扮演着关键的角色。近来人们越来越认识到先天免疫系统是哮喘发生的另一重要发病机制，其中在肺组织中巨噬细胞尤其是IL-4和IL-13 等诱导的M2型巨噬细胞在其中发挥关键作用。例如，研究显示与健康对照组相比，重症哮喘患者的BALF中的M2型巨噬细胞明显增多；在儿童哮喘中，也发现了增加的M2型巨噬细胞；此外，在尘螨诱发的小鼠过敏性哮喘模型中，M2型巨噬细胞的数量与气道炎症的严重程度呈现正相关；而将表达IL-4 受体的M2型巨噬细胞注入到小鼠中，能够明显加重肺部炎症反应。这些临床和小鼠模型研究都揭示M2型巨噬细胞和哮喘发生发展的相关性，因此抑制异常活化的M2型巨噬细胞，可以有效治疗和缓解哮喘疾病的发生和进行，然而人们目前对M2型巨噬细胞功能的调控并不是十分清楚。
近年来，人们发现长链非编码RNA的表达存在组织细胞特异性，且含量丰富，并可以通过染色质重塑和ceRNA海绵吸附等方式调控多个基因的表达。同时lncRNA的异常表达和功能失调在各种疾病中日益受到关注，相关的治疗策略也逐渐被探索。例如，胃癌中异常高表达的lcnRNA GClnc1就与肿瘤的大小、转移及预后不良密切相关。同时，更多的研究发现lncRNA也参与到调控巨噬细胞功能的过程中。例如，Ma等发现lincRNA-Tnfaip3作为 NF-κB的协同调节因子调节LPS诱导的炎症反应，从而参与M1型巨噬细胞极化的调控。但目前的研究多集中于lncRNA对M1型巨噬细胞极化调控的影响，对lncRNA在M2型巨噬细胞极化中的作用还知之甚少。而本发明通过RNA 高通量测序发现长链非编码RNA PTPRE-AS1在IL-4诱导的M2型巨噬细胞中异常高表达，而PTPRE-AS1又可以通过抑制IL-4诱导的MAPK/ERK1/2的信号通路激活，进一步地负向调控IL-4诱导的M2型巨噬细胞极化，形成一种独特的负反馈调节。更为重要的是，在过敏性哮喘小鼠模型中，证实 PTPRE-AS1敲除的小鼠其肺部炎症显著加重，揭示PTPRE-AS1调控的M2型巨噬细胞极化极有可能参与到小鼠哮喘的发生发展过程。与此同时，过敏性哮喘儿童PBMCs中PTPRE-AS1的异常低表达，也暗示其可能作为一种分子标记物用于哮喘的临床诊断与治疗。
综上，lncRNA作为一种新型调控分子，在人类多种疾病的发生和发展过程都有着重要作用。不同于以往的有或无的调节特点，lncRNA主要是在数量上对靶基因进行调节。这一特点是siRNA单靶点强大的调节作用或一些分子的拮抗剂和激动剂所无法比拟的。这展示了lncRNA在临床应用方面的巨大前景。本发明首次发现PTPRE-AS1，作为一种非编码RNA，参与负向调控IL-4诱导的M2型巨噬细胞极化过程，并且PTPRE-AS1敲除小鼠导致了更为严重的哮喘肺部炎症反应。因此PTPRE-AS1可能作为一个新的药物干预靶点，为治疗过敏性哮喘等疾病带来新的希望。
Claims (5)
1.一种LncRNA PTPRE-AS1在制备抑制蟑螂提取物诱导的过敏性哮喘药物中的用途。
2.根据权利要求1所述的一种LncRNA PTPRE-AS1在制备抑制蟑螂提取物诱导的过敏性哮喘药物中的用途，其特征在于：PTPRE-AS1参与负向调控IL-4诱导的M2型巨噬细胞极化。
3.根据权利要求2所述的一种LncRNA PTPRE-AS1在制备抑制蟑螂提取物诱导的过敏性哮喘药物中的用途，其特征在于：PTPRE-AS1通过显著降低ERK1/2的磷酸化进而抑制IL-4诱导的下游M2型巨噬细胞基因表达。
4.根据权利要求1所述的一种LncRNA PTPRE-AS1在制备抑制蟑螂提取物诱导的过敏性哮喘药物中的用途，其特征在于：PTPRE-AS1敲除导致小鼠的过敏性哮喘病情加重。
5.根据权利要求1所述的一种LncRNA PTPRE-AS1在制备抑制蟑螂提取物诱导的过敏性哮喘药物中的用途，其特征在于：PTPRE-AS1的表达在哮喘儿童的PBMCs中显著降低。
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