CN108144061A - microRNA-210抑制剂在制备治疗炎症性皮肤病药物上的应用 - Google Patents
microRNA-210抑制剂在制备治疗炎症性皮肤病药物上的应用 Download PDFInfo
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Abstract
本发明公开了一种microRNA‑210抑制剂在制备治疗炎症性皮肤病药物上的应用。本发明人通过大量的实验证明体外抑制microRNA‑210表达可显著增强其靶基因STAT6的表达，从而抑制角质形成细胞增殖及趋化因子CCL20的分泌，进一步抑制其趋化T细胞向皮损部位的迁移，同时，亦可抑制TH1及TH17的分化。microRNA‑210敲除及给予皮损内注射microRNA‑210抑制剂(胆固醇修饰的antagomiR‑210)特异性抑制microRNA‑210的表达，均可显著抑制小鼠皮肤炎症，改善T细胞免疫失衡。本发明为炎症性皮肤病提供了一个新的病理生理学机制，并提供了一种可用于制备治疗炎症性皮肤病药物的新策略。
Description
技术领域
本发明涉及一种内源性非编码小RNAs抑制剂的新医药用途，具体来说是一种microRNA-210抑制剂在制备治疗炎症性皮肤病药物中的应用，属于生物医学领域。
背景技术
炎症性皮肤病是一类常见的由免疫细胞和角质形成细胞共同介导的皮肤病。天然免疫系统的异常应答、T淋巴细胞的异常激活、各种炎症性细胞因子及其靶细胞(角质形成细胞)在炎症性皮肤病发病机制中起到重要作用。其中，辅助性T细胞(Th细胞)免疫失衡导致的一系列炎症反应在炎症性皮肤病中较为常见。Th细胞在一些抗原提呈细胞及天然免疫细胞的刺激下，各细胞亚群之间的免疫平衡发生紊乱，导致白介素、干扰素、肿瘤坏死因子等炎症细胞因子分泌异常，这些炎症细胞因子进一步作用于角质形成细胞，诱发皮肤屏障受损。许多炎症性皮肤病无论原因如何复杂，在病变发生机制上绝大部分都涉及了炎症和免疫，因此免疫抑制或免疫调节是这类疾病治疗的主要手段。然而几乎所有免疫抑制剂均有较为明显和严重的副作用。近年来，生物治疗取得了快速发展，许多单克隆抗体、免疫球蛋白以及专门针对免疫分子的生物制剂越来越多，虽然疗效肯定，但费用高昂，不良反应也不容忽视，长期使用的安全性尚不明确。因此，研发新型的疗效高、副作用小、成本低的免疫抑制或调节药物意义尤为重大。
MicroRNAs(miRNAs)是一种小的内源性非编码单链RNA分子，长约21-25个核苷酸，在进化上具有高度保守性、时序性和组织特异性，几乎存在于所有多细胞生物体中。这些小的miRNAs通常靶向一个或者多个mRNA，通过翻译水平的抑制或断裂靶mRNA而调控基因的表达，参与机体诸多生理病理过程。目前已有研究表明，多个miRNAs分子在皮肤中的表达比在其他器官中的表达具有显著差异性，如miR-203，miR-146a和miR-31，这些miRNAs分子或者与角质形成细胞的功能相关，或者在免疫细胞中特定表达。但是这些miRNAs分子是否可以应用于制备治疗炎症性皮肤病的药物尚不确定。本发明首先证明microRNA-210在咪喹莫特(IMQ)诱导的炎症性皮损中表达升高，其不仅调控角质形成细胞的增殖和趋化因子分泌，同时调控Th1及Th17细胞的分化，然后进一步通过体外细胞实验和体内动物实验揭示以microRNA-210为作用靶点的抑制剂可明显缓解IMQ诱导的皮肤炎症的发生和严重程度。我们的结果提示microRNA-210抑制剂可以应用于制备治疗炎症性皮肤病的药物，在炎症性皮肤病患者皮损治疗方面具有巨大的应用前景。
发明内容
本发明的第一个目的在于提供microRNA-210抑制剂在制备治疗炎症性皮肤病的药物中的应用。所述的这种应用为治疗炎症性皮肤病提供了一种新的途径，并改善了治疗效果。
microRNA-210的抑制剂在制备治疗炎症性皮肤病药物上的应用，microRNA-210的序列如SEQ NO.1所示。
所述炎症性皮肤病包括：银屑病、特应性皮炎、湿疹或副银屑病。
所述microRNA-210抑制剂选自能降低microRNA-210表达含量的小干扰RNA、dsRNA、shRNA、微小RNA、反义核苷酸；或者能表达或形成所述小干扰RNA、dsRNA、shRNA、微小RNA、反义核苷酸的构建物。
所述microRNA-210抑制剂优选购买自广州锐博生物科技有限公司，产品名称为micrOFFTM mmu-miR-210-3p antagomir。
所述microRNA-210抑制剂的核苷酸序列为5’-ucagccgcugugucacacgcacag-3’,并且具有3’-胆固醇及5’-OMe修饰，修饰后的microRNA-210抑制剂保持所述microRNA-210抑制剂的活性。
所述microRNA-210抑制剂的应用方法为皮损局部用药。
本发明的第二个目的是提供microRNA-210的抑制剂在制备抑制IL-17A、IL-17F和IFN-γ分泌，促进IL-4分泌的制剂上的应用；microRNA-210的序列如SEQ NO.1所示。
本发明的第三个目的是提供microRNA-210的抑制剂在制备增强靶基因STAT6表达的制剂上的应用；microRNA-210的序列如SEQNO.1所示。
本发明的第四个目的是提供microRNA-210的抑制剂在制备抑制角质形成细胞增殖及趋化因子CCL20的分泌，进一步抑制其趋化T细胞向皮损部位的迁移的制剂上的应用；microRNA-210的序列如SEQ NO.1所示。
本发明的第五个目的是提供microRNA-210的抑制剂在制备抑制TH1及TH17的分化，促进TH2细胞分化的制剂上的应用；microRNA-210的序列如SEQ NO.1所示。
本发明人通过大量的实验证明在IMQ诱导的小鼠炎症性皮损中microRNA-210表达显著上调，体外抑制microRNA-210表达可显著增强其靶基因STAT6的表达，从而抑制角质形成细胞增殖及趋化因子CCL20的分泌，进一步抑制其趋化T细胞向皮损部位的迁移，同时，亦可抑制TH1及TH17的分化，促进TH2细胞分化。在IMQ诱导的小鼠炎症性皮损中过表达microRNA-210(给予microRNA-210模拟物agomiR-210皮内注射)，可明显促进并加重小鼠皮肤的炎症反应，促进皮损中TH1及TH17细胞浸润。而microRNA-210敲除及给予皮损内注射microRNA-210抑制剂(胆固醇修饰的antagomiR-210)特异性抑制microRNA-210的表达，均可显著抑制小鼠皮肤炎症，改善T细胞免疫失衡。
附图说明
为了更清楚地说明本发明中的技术方案，下面将对本发明描述中所需要使用的附图作简单地介绍。
图1A显示了IMQ诱导的小鼠炎症性皮损及正常对照小鼠的外观及HE染色病理切片(放大倍数100倍)；
图1B显示了IMQ诱导的小鼠炎症性皮损棘皮症量化结果；
图1C显示了IMQ诱导的小鼠炎症性皮损炎症细胞浸润量化结果；
图1D显示了IMQ诱导的小鼠炎症性皮损及正常小鼠皮肤中miR-210的表达水平；
图1E显示了IMQ诱导的小鼠炎症性皮损及正常小鼠皮肤中miR-210原位杂交；
图2A显示了24h时，过表达或抑制microRNA-210对角质形成细胞增殖的影响；
图2B显示了48h时，过表达或抑制microRNA-210对角质形成细胞增殖的影响；
图2C显示了microRNA-210调控角质形成细胞增殖的变化趋势；
图3A显示了过表达microRNA-210对角质形成细胞分泌趋化因子及炎症因子的影响；
图3B显示了抑制microRNA-210表达对角质形成细胞分泌趋化因子及炎症因子的影响；
图3C显示了过表达或抑制microRNA-210对角质形成细胞培养上清中CCL20分泌水平的影响；
图3D-E显示了在角质形成细胞中过表达或抑制microRNA-210对T细胞趋化的影响；
图4A显示了 CD4+T细胞向不同T细胞亚群分化过程中，microRNA-210的表达改变；
图4B显示了过表达microRNA-210对不同T细胞亚群分化的影响；
图4C显示了抑制microRNA-210表达对不同T细胞亚群分化的影响；
图5A显示了agomiR-210或antagomiR-210可显著促进或抑制microRNA-210在CD4+T细胞中的表达；
图5B,C显示了过表达或抑制microRNA-210对CD4+T细胞培养上清中细胞因子蛋白水平的影响；
图5D,E显示了过表达或抑制microRNA-210对CD4+T细胞中细胞因子mRNA水平的影响；
图6显示了microRNA-210调控下游靶基因STAT6表达；
图7显示了皮损内过表达microRNA-210可显著促进并加重IMQ诱导的小鼠炎症性皮损；
图8显示了特异性敲除microRNA-210可显著减轻IMQ诱导的小鼠炎症性皮损；
图9显示了皮损内注射antagomiR-210可显著改善IMQ诱导的小鼠炎症性皮损；
图10显示了皮损内注射antagomiR-210可显著改善IMQ诱导的小鼠脾脏细胞中T细胞免疫紊乱。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明实施方式作进一步地详细描述。应该理解的是，这些实施例仅用于例证的目的，决不限制本发明的保护范围。
实施例1、microRNA-210在IMQ诱导的小鼠炎症皮损中表达升高
选取12只7周龄大小、健康雌性Balb/c小鼠，背部脱毛2×2cm2后随机分为两组：第一组为模型组，每只小鼠每日背部脱毛区外涂5％咪喹莫特软膏62.5mg/d，连续6天；第二组为正常组，每只小鼠每日背部脱毛区外涂基质软膏62.5mg/d，连续6天。造模第7天，采用断颈法处死各组小鼠，取其背部皮损，放入福尔马林溶液中固定过夜，脱水、石蜡包埋，切片行HE染色，镜下观察组织学病理改变。结果发现：与正常组小鼠相比，模型组小鼠皮肤出现肉眼可见的鳞屑性红斑，表皮明显增厚，角化过度及角化不全，表皮突延长，棘层肥厚、颗粒层消失，真皮层炎细胞浸润及血管扩张明显(图1A-C)。证明IMQ诱导的小鼠炎症性皮肤病造模成功。
取IMQ诱导的小鼠炎症皮损区及正常小鼠皮肤组织，采用Trizol法提取组织totalRNA，逆转录后应用real-time PCR检测microRNA-210的表达。同时，采用原位杂交技术检测microRNA-210在IMQ诱导的小鼠炎症皮损区中的表达及分布。结果发现：与正常皮肤相比，IMQ诱导的小鼠炎症皮损中microRNA-210的表达显著升高(图1D)；原位杂交结果亦显示，microRNA-210的表达在IMQ小鼠表皮区明显增强(图1E)。
图1(A)显示IMQ诱导的小鼠炎症性皮损。图1(B)显示IMQ小鼠(n＝6)与正常小鼠(n＝6)皮损中表皮厚度情况，***P<0.001。图1(C)显示IMQ小鼠(n＝6)与正常小鼠(n＝6)皮损中炎细胞浸润情况，***P<0.001。图1(D)显示IMQ小鼠(n＝6)与正常小鼠(n＝6)皮损中microRNA-210的表达情况，***P<0.001。图1(E)显示microRNA-210在IMQ小鼠皮损组织与正常小鼠皮肤中的表达与分布。采用双侧t检验进行统计学分析。
实施例2、microRNA-210异常表达对角质形成细胞及T细胞的影响及机制研究
1)microRNA-210促进角质形成细胞增殖
本实验采用人原代角质形成细胞。转染12小时(hours,hrs)前，将人原代角质形成细胞接种于96孔板，用相应的100μL无血清培养基于37℃、5％CO2培养箱中培养12h。当细胞生长至90％融合度时，向角质形成细胞中转染agomiR-210(microRNA-210的模拟物)、antagomiR-210以及相应的阴性对照，6hrs后更换新鲜培养基，继续培养24hrs或48hrs。在培养结束前4hrs，向各孔中加入10μL CCK8溶液继续培养4hrs。于450nm波长处检测细胞增殖情况。结果显示：过表达microRNA-210可以显著促进人原代角质形成细胞增殖；相反地，抑制microRNA-210的表达则显著抑制角质形成细胞增殖(图2A-C)。
图2(A)显示24h时microRNA-210对角质形成细胞增殖的影响，*P<0.05，**P<0.01。图2(B)显示48h时microRNA-210对角质形成细胞增殖的影响，**P<0.01。图2(C)显示microRNA-210调控角质形成细胞增殖的变化趋势。采用双侧t检验进行统计学分析。
2)microRNA-210促进角质形成细胞分泌趋化因子CCL20进而影响T细胞迁移
本实验采用人原代角质形成细胞。转染12hrs前，将人原代角质形成细胞接种装有1ml无血清培养基的24孔板，于37℃、5％CO2培养箱中培养12h。当细胞生长至90％融合度时，向角质形成细胞中转染agomiR-210(microRNA-210的模拟物)、antagomiR-210以及相应的阴性对照，6hrs后更换新鲜培养基，继续培养48hrs。收集细胞，提取total RNA，real-time PCR检测细胞因子及趋化因子表达水平。结果发现，过表达microRNA-210可显著促进角质形成细胞分泌CCL20(图3A)，而抑制microRNA-210的表达则明显抑制CCL20的分泌(图3B)。
同样的方法培养细胞，收集细胞上清，一部分用于ELISA检测趋化因子CCL20蛋白分泌水平，结果与上述PCR结果相一致(图3C)；另一部分用于接下来的趋化实验。5.0μm孔径大小的趋化小室用于该趋化实验。磁珠分离正常人外周血CD4+T细胞，用200μL无血清培养基重悬5×105个CD4+T细胞接种于上层小室，600μL角质形成细胞培养上清接种于下层小室，于37℃、5％CO2培养箱中培养90min。用棉签将上层小室未趋化的细胞轻轻刮除，90％酒精固定滤网下层表面的趋化细胞15min，然后用0.1％的结晶紫染色10min，PBS漂洗两次，显微镜下计数5个不同视野下的趋化细胞数目，取其平均值。结果显示，过表达microRNA-210的角质形成细胞分泌的上清可显著促进T细胞迁移，而抑制microRNA-210的角质形成细胞分泌的上清则可显著抑制T细胞迁移(图3D,E)。
图3(A)显示过表达microRNA-210对角质形成细胞趋化因子和细胞因子分泌的影响，*P<0.05。图3(B)显示抑制microRNA-210对角质形成细胞趋化因子和细胞因子分泌的影响，*P<0.05。图3(C)显示过表达或抑制microRNA-210对角质形成细胞分泌趋化因子CCL20蛋白水平的影响，**P<0.01，***P<0.001。图3(D，E)显示过表达或抑制microRNA-210对角质形成细胞趋化T细胞能力的影响，***P<0.001。采用双侧t检验进行统计学分析。
3)microRNA-210促进TH1及TH17细胞分化，抑制TH2细胞分化
收集正常人外周血，磁珠分选 CD4+T细胞，首先检测CD4+T细胞向不同T细胞亚群分化过程中，miR-210的表达改变；然后向 CD4+T中分别转染agomiR-210、antagomiR-210及相应对照，在体外诱导分化条件下，诱导 CD4+T细胞向TH1、TH2、TH17及iTreg方向分化。5天后，采用流式细胞术检测各实验组中不同T细胞亚群的分化百分比。结果显示：microRNA-210表达在TH17细胞体外诱导分化过程中增加最为显著(图4A)；过表达microRNA-210可显著促进 CD4+T细胞向TH1及TH17分化，抑制TH2分化(图4B)；而抑制microRNA-210表达则可抑制 CD4+T细胞向TH1及TH17分化，促进TH2分化(图4C)；microRNA-210对体外Treg诱导分化无显著影响(图4B,C)。
图4(A)显示 CD4+T细胞向不同T细胞亚群分化过程中，microRNA-210的表达改变，***P<0.001，NS：无统计学意义，采用单因素方差分析进行统计学分析。图4(B)显示过表达microRNA-210对不同T细胞亚群分化的影响。图4(C)显示抑制microRNA-210对不同T细胞亚群分化的影响。
4)microRNA-210促进CD4+T细胞中IL-17A、IL-17F和IFN-γ的分泌，抑制IL-4的分泌
我们向正常人外周血CD4+T细胞中转染agomiR-210、antagomiR-210及其相应阴性对照，48h后，收集细胞上清及细胞，通过ELISA及real-time PCR方法，分别检测转染效率、细胞因子蛋白及RNA水平改变。结果显示：agomiR-210或antagomiR-210可显著促进或抑制CD4+T细胞中microRNA-210表达(图5A)；过表达microRNA-210可明显促进IL-17A、IL-17F和IFN-γ的分泌，抑制IL-4的分泌(图5B,D)，而抑制microRNA-210则可显著抑制IL-17A、IL-17F和IFN-γ的分泌，促进IL-4的分泌(图5C,E)。
图5(A)显示agomiR-210或antagomiR-210可显著促进或抑制CD4+T细胞中microRNA-210表达，**P<0.01。图5(B,C)显示过表达或抑制microRNA-210对CD4+T细胞培养上清中细胞因子蛋白水平的影响，*P<0.05，**P<0.01，***P<0.001。图5(D,E)显示过表达或抑制microRNA-210对CD4+T细胞中细胞因子mRNA水平的影响，*P<0.05，**P<0.01，***P<0.001，NS：无统计学意义。采用双侧t检验进行统计学分析。
5)microRNA-210的下游靶基因筛选及验证
我们首先通过TargetScan,miRWalk,miRanda和RNA22等生物信息学软件预测发现STAT6可能为microRNA-210下游靶基因，且与T细胞分化密切相关(图6A)。其次我们进一步在HEK293T细胞中进行荧光素酶报告基因实验验证，发现过表达microRNA-210可显著抑制转染野生型质粒(STAT6WT)的HEK293T细胞中的荧光素酶活性，而对转染STAT6结合位点突变质粒(STAT6MU)的HEK293T细胞中的荧光素酶活性无影响(图6B)。然后，我们在人CD4+T细胞中进行了microRNA-210过表达及干扰实验证明microRNA-210可调控STAT6的蛋白表达水平(图6C)。此外，我们还应用western blot技术检测了IMQ诱导的小鼠模型皮损中靶基因STAT6的蛋白水平，发现与正常小鼠相比，STAT6蛋白表达水平在IMQ小鼠皮损中显著下调(图6D)。
图6(A)显示靶基因STAT6的mRNA 3’非编码区与microRNA-210的结合位点序列及突变后结合位点序列(红色区域)。图6(B)显示荧光素酶报告基因实验显示STAT6的mRNA 3’非编码区是microRNA-210的直接作用靶点，*P<0.05。图6(C)显示microRNA-210可以调控STAT6蛋白表达水平。图6(D)显示IMQ小鼠皮损组织(n＝3)与正常小鼠皮肤组织(n＝3)中STAT6的蛋白表达水平，*P<0.05。采用双侧t检验进行统计学分析。
本实施例证明，microRNA-210可直接调控靶基因STAT6的表达。抑制microRNA-210可显著抑制角质形成细胞增殖及趋化因子CCL20的分泌，进一步阻止其趋化炎症性T细胞向皮损部位迁移。同时，抑制microRNA-210可显著抑制TH1及TH17细胞的分化，促进TH2细胞分化。
实施例3、体内过表达microRNA-210可显著促进并加重IMQ诱导的小鼠炎症性皮损
选取6-8周龄健康雌性Balb/c小鼠，背部脱毛2×2cm，随机分为两组：1)agomiR-210组：每日背部外涂5％IMQ软膏62.5mg/d，连续6天，并于第0，1，2，3天背部皮内注射agomiR-210 150μl(5nmol)；2)agomiR-NC组：每日背部外涂5％IMQ软膏62.5mg/d，连续6天，并于第0，1，2，3天背部皮内注射agomiR-NC 150μl(5nmol)。分别于造模第4天、第7天、第10天及第14天各组处死3只小鼠，观察皮损临床及病理改变，并进行组织学分析，采用real-timePCR检测microRNA-210的表达改变。结果显示：在造模第4天及第7天，microRNA-210表达在agomiR-210组显著增高(图7A)。皮内注射agomiR-210可显著促进并加重IMQ诱导的小鼠炎症性皮损改变，agomiR-210组小鼠脾肿大较agomiR-NC组小鼠亦明显加重，以上指标在第7天达高峰，且差异最为明显；在疾病恢复期(8-14天)两组临床及病理改变无显著差异(图7B-F)。
图7(A)显示agomiR-210注射过程中，microRNA-210的表达改变，***P<0.001。图7(B)显示第四天、第七天、第十天及第十四天，agomiR-210组和agomiR-NC组小鼠外观和HE染色病理改变。图7(C)显示第七天，agomiR-210组和agomiR-NC组小鼠脾肿大的情况。图7(D)显示第七天，agomiR-210组(n＝6)和agomiR-NC组(n＝6)小鼠皮损中表皮增厚情况，**P<0.01。图7(E)显示第七天，agomiR-210组(n＝6)和agomiR-NC组(n＝6)小鼠皮损中炎细胞浸润情况，**P<0.01。图7(F)显示第七天，agomiR-210组和agomiR-NC组小鼠皮损真皮单细胞悬液中TH1和TH17细胞比例改变。采用双侧t检验进行统计学分析。
实施例4、特异性microRNA-210敲除抑制IMQ诱导的小鼠炎症性皮损
构建C57BL/6J背景的microRNA-210全敲小鼠(miR-210KO小鼠)，选取6-8周龄的KO小鼠及WT小鼠，每日背部脱毛区外涂5％IMQ软膏62.5mg/d，连续6天，于第7天处死各组小鼠，进行组织病理分析及脾脏T细胞亚群比例检测。结果显示：microRNA-210敲除可显著抑制IMQ诱导的小鼠炎症性皮损，改善脾脏T细胞免疫紊乱(图8)。
图8(A)显示IMQ诱导的miR-210KO小鼠及WT小鼠的炎症性皮损外观改变及HE病理图片。图8(B)显示IMQ诱导的KO小鼠及WT小鼠脾肿大情况。图8(C)显示IMQ诱导的KO小鼠(n＝5)及WT小鼠(n＝5)皮损中表皮增厚情况，***P<0.001。图8(D)显示IMQ诱导的KO小鼠(n＝5)及WT小鼠(n＝5)皮损中炎细胞浸润情况，**P<0.01。图8(E)显示IMQ诱导的KO小鼠及WT小鼠脾脏中T细胞免疫紊乱情况，microRNA-210敲除可显著降低IMQ小鼠脾脏细胞中TH1及TH17比例，增高TH2细胞比例。采用双侧t检验进行统计学分析。
实施例5、皮损内注射microRNA-210抑制剂可显著改善小鼠炎症性皮损
选取12只6周龄大小、健康雌性Balb/c小鼠，利用胆固醇修饰的antagomiR-210(将胆固醇连接在antagomiR-210的5’端，增加其跨膜性，可特异性抑制microRNA-210表达)经背部脱毛区分三点皮内注射。实验分为两组：(1)antagomiR-210组：每日背部外涂5％IMQ软膏62.5mg/d，连续6天，并于第0，1，2，3天背部皮内注射antagomiR-210 150μl；(2)antagomiR-NC组：每日背部外涂5％IMQ软膏62.5mg/d，连续6天，并于第0，1，2，3天背部皮内注射antagomiR-NC 150μl。1周后取背部皮损，一部分用福尔马林溶液固定过夜、脱水、石蜡包埋，切片行HE染色观察组织病理改变，另一部分提取组织RNA，real-time PCR检测microRNA-210的表达。结果显示：注射胆固醇修饰的antagomiR-210可显著抑制皮损中microRNA-210的表达，且明显改善小鼠炎症性皮损(图9A,B)；组织病理学上antagomiR-210可显著抑制表皮增厚、表皮突延长及真皮炎症细胞浸润(图9C-E)，显著改善IMQ诱导的小鼠炎症性皮损的病理改变。同时，皮内注射antagomiR-210亦可显著改善IMQ诱导的小鼠脾脏中T细胞免疫紊乱(图10)。
图9(A)显示antagomiR-210组(n＝5)和antagomiR-NC组(n＝5)小鼠皮损中microRNA-210的表达情况，***P<0.001。图9(B)显示antagomiR-210组和antagomiR-NC组小鼠炎症性皮损的外观表现。图9(C)显示antagomiR-210组和antagomiR-NC组小鼠炎症性皮损的病理改变。图9(D)显示antagomiR-210组(n＝5)和antagomiR-NC组(n＝5)小鼠皮损表皮增厚的情况，*P<0.05。图9(E)显示antagomiR-210组(n＝5)和antagomiR-NC组(n＝5)小鼠皮损中炎细胞浸润情况，***P<0.001。图10显示antagomiR-210组和antagomiR-NC组小鼠脾脏中T细胞免疫紊乱情况，皮内注射antagomiR-210可明显减少IMQ小鼠脾脏细胞中TH1及TH17比例，增高TH2细胞比例。采用双侧t检验进行统计学分析。
以上所述仅为本发明的优选实施例，对本发明而言仅是说明性的，而非限制性的；本领域普通技术人员理解，在本发明权利要求所限定的原则和范围内可对其进行许多改变，修改，甚至等效变更，但都将落入本发明的保护范围内。
序列表
<110> 中南大学湘雅二医院
<120> microRNA-210抑制剂在制备治疗炎症性皮肤病药物上的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> RNA
<213> 智人(Homo sapiens)
<400> 1
agucggcgac agugugcgug uc 22
<210> 2
<211> 24
<212> RNA
<213> 未知(Unknown)
<400> 2
ucagccgcug ugucacacgc acag 24
Claims (10)
1.microRNA-210的抑制剂在制备治疗炎症性皮肤病药物上的应用，microRNA-210的序列如SEQ NO.1所示。
2.根据权利要求1所述的应用，其特征在于，所述炎症性皮肤病包括：银屑病、特应性皮炎、湿疹或副银屑病。
3.根据权利要求2所述的应用，其特征在于，所述microRNA-210抑制剂选自能降低microRNA-210表达含量的小干扰RNA、dsRNA、shRNA、微小RNA、反义核苷酸；或者能表达或形成所述小干扰RNA、dsRNA、shRNA、微小RNA、反义核苷酸的构建物。
4.根据权利要求3所述的应用，其特征在于，所述microRNA-210抑制剂购买自广州锐博生物科技有限公司，产品名称为micrOFFTM mmu-miR-210-3p antagomir。
5.根据权利要求4所述的应用，其特征在于，所述microRNA-210抑制剂的核苷酸序列为5’-ucagccgcugugucacacgcacag-3’,并且具有3’-胆固醇及5’-OMe修饰，修饰后的microRNA-210抑制剂保持所述microRNA-210抑制剂的活性。
6.根据权利要求5所述的应用，其特征在于，所述microRNA-210抑制剂的应用方法为皮损局部用药。
7.microRNA-210的抑制剂在制备抑制IL-17A、IL-17F和IFN-γ分泌，促进IL-4分泌制剂上的应用；microRNA-210的序列如SEQ NO.1所示。
8.microRNA-210的抑制剂在制备增强靶基因STAT6表达制剂上的应用；microRNA-210的序列如SEQ NO.1所示。
9.microRNA-210的抑制剂在制备抑制角质形成细胞增殖及趋化因子CCL20的分泌，进一步抑制其趋化T细胞向皮损部位的迁移制剂上的应用；microRNA-210的序列如SEQ NO.1所示。
10.microRNA-210的抑制剂在制备抑制TH1及TH17的分化，促进TH2细胞分化制剂上的应用；microRNA-210的序列如SEQ NO.1所示。
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| WO2019174413A1 (zh) * | 2018-03-13 | 2019-09-19 | 中南大学湘雅二医院 | microRNA-210抑制剂在制备治疗炎症性皮肤病药物上的应用 |
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| WO2010005850A1 (en) | 2008-07-08 | 2010-01-14 | The J. David Gladstone Institutes | Methods and compositions for modulating angiogenesis |
| CN105617398A (zh) * | 2014-10-27 | 2016-06-01 | 中国人民解放军第二军医大学 | microRNA-342-3p和microRNA-210及其抑制剂的用途 |
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| US11542502B2 (en) | 2018-03-13 | 2023-01-03 | The Second Xiangya Hospital Of Central South University | Application of microRNA-210 inhibitor in preparation of drugs for treating inflammatory skin diseases |
| CN108690123A (zh) * | 2018-06-21 | 2018-10-23 | 上海交通大学医学院 | 短肽在制备免疫调节药物中的应用 |
| CN108690123B (zh) * | 2018-06-21 | 2021-11-19 | 上海交通大学医学院 | 短肽在制备免疫调节药物中的应用 |
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