CN107019693A - α‑倒捻子素在制备用于治疗自身免疫疾病的药物中的应用 - Google Patents
α‑倒捻子素在制备用于治疗自身免疫疾病的药物中的应用 Download PDFInfo
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Abstract
本发明公开了α‑倒捻子素在制备用于治疗自身免疫疾病的药物中的应用，本发明研究发现α‑倒捻子素作为Th17细胞活性的抑制剂，可以抑制Th17细胞分化、形成。
Description
技术领域
本发明涉及α-倒捻子素的新用途，尤其涉及α-倒捻子素在制备用于治疗自身免疫疾病的药物中的应用。
背景技术
约5％人口患有自身免疫疾病，大约70多种人类疾病都和自免疫失调有关(Goodnow et al.,2005)。目前自身免疫疾病的治疗主要依赖于一些非选择性的免疫抑制剂，其疗效有限且副作用大，临床中还没有非常特效的治疗自身免疫疾病的药物。因此，临床上急需开发一种新型的高疗效且低副作用的自免疫药物。
最新研究表明一种新的T细胞亚群Th17细胞和人类自身免疫疾病和相关动物模型的发生密切相关(Kikly et al.,2006)。在风湿性关节炎、牛皮癣、哮喘、系统性红斑狼疮、特征性皮炎、炎症性肠炎等自身免疫疾病中，Th17细胞的特征细胞因子IL17A表达上调，而抑制IL17A表达和Th17细胞分化可以减轻自身免疫疾病的发生或临床严重程度(Bowman etal.,2006；Kikly et al.,2006)。现有研究表明Th17细胞的分化形成受到转录因子RORγt的控制，而在RORγt基因敲除的小鼠中，Th17细胞的分化能力下降，数量减少，同时诱导发生的小鼠EAE自身免疫疾病概率和临床打分指标均有显著的降低(Ivanov et al.,2006)。这意味着RORγt的功能抑制剂可以作为开发Th17介导的自身免疫疾病治疗药物的定向靶标。
RORγt是类固醇类核受体家族成员，蛋白分子中包括一个保守的DNA结合域和有12个螺旋构成的配体结合域，而配体结合域是结合配体、核定位和二聚体形成的重要区域。类固醇类受体共激活分子SRCs能够通过结合配体结构域的AF2区域来解聚转录抑制复合物，招募转录激活分子，启动相关基因转录(Glass and Rosenfeld,2000)。目前，RORγt的天然配体还没有找到，但最近两个研究发现人工合成分子地高辛及其衍生物(Huh et al.,20011；Fujita-Sato et al.,2011)和SR1001(Solt et al.,2011)能够特异性地结合到RORγt的配体结构域，抑制RORγt的功能并降低Th17细胞的分化能力，减弱小鼠自身免疫疾病EAE的临床症状。然而，地高辛及其衍生物具有很强的毒性(Paula et al.,2005)；SR1001分子尽管在体外能够有效抑制Th17细胞的分化，但在体内实验中高浓度给药下(40mg/kg)，也只能略微减轻EAE的临床症状。此外，SR1001能够同其他的RORs作用，选择性较差(Solt etal.,2011)。这些研究一方面表明抑制RORγt的功能来治疗自身免疫疾病是一个可行的方案，但目前还没有找到一个理想的靶标药物。因此寻找高效低毒且具有较强特异性的RORγt功能抑制剂将是开发Th17介导的自身免疫疾病药物的一个重要方向。
发明内容
本发明目的在于克服现有技术存在的不足，而提供α-倒捻子素在制备用于治疗自身免疫疾病的药物中的应用。为实现上述目的，本发明采取的技术方案为：α-倒捻子素在制备用于治疗自身免疫疾病的药物中的应用，所述α-倒捻子素的结构式为：
作为上述技术方案的改进，所述自身免疫疾病为Th17细胞介导的自身免疫疾病。
作为上述技术方案的改进，所述自身免疫疾病包括牛皮癣、多发性硬发症、风湿性关节炎、系统性红斑狼疮、特征性皮炎、哮喘和炎症性肠道病。
另外，本发明还提供一种药物组合物，所述药物组合物包括α-倒捻子素和药学上可接受的载体，所述α-倒捻子素的结构式为：
在上述技术方案中，“药学上可接受的载体”包括任何和所有生理上相容的溶剂、分散介质、包衣料、抗细菌和抗真菌剂、等渗和吸收延缓剂等。药学上可接受的载体的实例包括水、盐水、磷酸缓冲盐水、右旋糖酐、甘油、乙醇等中的一个或多个以及它们的组合。在很多情况下，优选的是将等渗剂例如糖、多元醇或氯化钠包括在组合物中。药学上可接受的载体还可包含少量的能提高抗体或抗体部分的货架期或有效性的辅助物质，如湿润剂或乳化剂、防腐剂或缓冲液。
作为上述技术方案的改进，所述药物组合物的剂型是注射液或冻干制剂。
在上述技术方案中，本发明的药物组合物可以为多种形式。这些形式包括例如液体、半固体和固体剂型，如液体溶液剂(例如可注射和可输注溶液剂)、分散剂或混悬剂、片剂、丸剂、散剂、脂质体和栓剂。优选的形式取决于预定的给药方式和治疗应用。典型的优选组合物为可注射或可输注溶液剂形式。优选的给药方式是胃肠外(例如静脉内、皮下、腹膜内、肌肉内)给药。在一个优选的实施方案中，药物组合物通过腹膜注射给予。还可将辅助性活性化合物掺入到组合物中。
另外，本发明还提供所述药物组合物在制备用于治疗自身免疫疾病的药物中的应用。
作为上述技术方案的改进，所述自身免疫疾病为Th17细胞介导的自身免疫疾病。
作为上述技术方案的改进，所述自身免疫疾病包括牛皮癣、多发性硬发症、风湿性关节炎、系统性红斑狼疮、特征性皮炎、哮喘和炎症性肠道病。
本发明的有益效果在于：本发明提供α-倒捻子素在制备用于治疗自身免疫疾病的药物中的应用，本发明利用基于转录因子活性的荧光素酶活性筛选系统，通过筛选获得活性分子α-倒捻子素，α-倒捻子素具有抑制Th17细胞分化的关键转录因子RORγt的表达；体外Th17细胞分化实验验证这些化合物具有抑制Th17分化的功能，能够抑制RORγt的靶基因IL17A和IL17F分子的转录表达；用流式细胞仪检测细胞内的IL17A表达也得到明显抑制；证实α-倒捻子素具有明显抑制Th17细胞活性的功能；α-倒捻子素作为Th17细胞活性的抑制剂，可以用于自身免疫疾病的治疗中。
附图说明
图1为α-倒捻子素对RORγt表达的抑制率的曲线图；图中α-倒捻子素的简写为α-D，以下相同；
图2为α-倒捻子素抑制Th17细胞体外诱导分化过程中IL17A的mRNA转录的柱状图；
图3显示α-倒捻子素抑制IL-17A蛋白表达。
具体实施方式
为更好地说明本发明的目的、技术方案和优点，下面将结合具体实施例、和附图对本发明作进一步说明。
试剂来源
细胞系Jurkat为本实验室保存；DH5α细菌菌株由北京中科院遗传所马润林教授馈赠；限制性内切酶购自美国fermentas公司；DNA连接酶购自美国NEB公司；报告基因序列IRES-GFP为发明人自有保存(也可以采用其他现有的报告基因序列)；报告质粒pGL4.31[luc2P/GAL4UAS/Hygro]和pBIND质粒购自美国Promega公司；DMEM培养基和RPMI1640培养基以及培养细胞用的丙酮酸钠、谷氨酰胺、β巯基乙醇、非必须氨基酸、双抗是购自美国Life公司；胎牛血清(FBS)购自美国Hyclone公司；荧光素酶报告基因检测试剂盒购自美国Promega公司；Lipo2000购自美国Life公司；无内毒素质粒提取试剂盒购自Sigma公司；8～12周龄的C57小鼠购自中山大学实验动物中心(SPF级)；T细胞电转试剂盒和电穿孔仪购自瑞士Lonza公司；潮霉素B购自瑞士Roche公司；逆转录试剂盒购自大连Takara公司；Realtime检测试剂盒购自美国Promega公司；细胞因子hTGF-β和mIL-6购自美国RD公司；小鼠CD3、CD4、IL17A和CD28抗体购自美国eBioscience公司；其他常用化学试剂购自Sigma公司和上海生工公司；本发明所采用的α-倒捻子素可购买，也可以自行制备。
α-倒捻子素的制备方法
山竹果皮100g，粉碎，5倍量95％乙醇回流提取3次，每次1小时；合并提取液，减压浓缩得浸膏9.6g,将此浸膏悬浮于100ml水中，依次以等体积的石油醚、氯仿、乙酸乙酯、正丁醇萃取萃取，每种溶剂萃取3次；合并氯仿萃取部分，浓缩后得浸膏3.2g，经硅胶柱层析(200～300目)，乙酸乙酯/石油醚洗脱，得黄色固体160mg。
质粒构建
将荧光蛋白基因IRES-GFP目的片段通过Not1单酶切位点插入到pBIND载体中完成pBIND-IRES-GFP的质粒构建；用PCR法从PBMC(外周血单个核细胞)的cDNA中调取人的RORγt基因(hRORγt)，hRORγt调取时两端加上BamH1的单酶切位点；再将hRORγt基因克隆到pBIND-IRES-GFP质粒中，完成pBIND-Gal4DBD-hRORγt-IRES-GFP重组质粒的构建(Gal4DBD的序列是pBIND质粒自带的)。
细胞培养
Jurkat细胞培养在含10％FBS(胎牛血清)，1％双抗，2mM谷氨酰胺，1mM丙酮酸钠，50μM的β巯基乙醇的RPMI1640完全培养基中。细胞置于5％CO2，37℃培养箱中恒温培养，约每两天传代一次。
pGL4.31+hRORγt+Jurkat稳定细胞系的构建
将20μg经Not1单酶切的质粒pGL4.31[luc2P/GAL4UAS/Hygro]电转入Jurkat细胞(1×107)中，再用200μg/ml的潮霉素B进行抗性筛选；筛选进行2～3周后，再将20μg用限制性内切酶Eam1105酶切的pBIND-Gal4DBD-hRORγt-IRES-GFP重组质粒电转至经潮霉素B筛选后的Jurkat细胞中，再次转染后的细胞继续培养2～3周，之后用GFP荧光蛋白标记进行流式分选，并收集GFP+阳性Jurkat细胞。
EC50的测定
EC50的测定基于构建的Gal4/hRORγt的报告体系：将pGL4.31+hRORγt+Jurkat细胞铺板至96孔圆底板中，每孔2×104个细胞，5％CO2，37℃培养箱中恒温培养过夜，然后按浓度梯度设置不同浓度值，在不同浓度的α-倒捻子素下作用6h，之后收集细胞，进行luciferase活性检测以得到α-倒捻子素的EC50的值；结果如图1所示。
如图1所示，α-倒捻子素具有抑制Th17细胞分化的关键转录因子RORγt的表达，根据计算公式测得α-倒捻子素抑制RORγt转录因子表达的EC50为0.5312μM。
α-倒捻子素对Th17细胞的体外分化的影响
实验前一天晚上用含有5μg/ml CD3抗体、1μg/ml CD28抗体的PBS溶液包被六孔板，每孔1ml，4℃包被过夜。第二天开展实验，首先用Miltenyi磁珠分选小鼠脾脏的CD4+T细胞，分选后的细胞按1×106个/ml的细胞密度重悬细胞，向包被后的六孔板的每个孔加2ml的细胞悬液，即每孔细胞数为2×106个，激活24h。24h后，收集六孔板中的细胞至50ml离心管中，再向溶液中加入终浓度为5ng/ml的hTGF-β和30ng/ml的mIL-6的细胞因子，混匀，体外诱导CD4+T细胞向Th17细胞分化。混匀后的细胞悬液，按每孔100μl分配至96孔板中，再向每孔加1μl的终浓度为1μMα-倒捻子素(并设置3个孔的DMSO对照组)，37℃、5％二氧化碳培养48h。48h后，每孔补加100μl RPMI1640完全培养基以及终浓度为5ng/ml的hTGF-β、30ng/ml的mIL-6的细胞因子和1μl的终浓度为5μMα-倒捻子素，补加完成后再培养48h。至此，CD4+T细胞体外诱导Th17分化培养了约5天，5天后收集细胞，用于抽提RNA和细胞染色后的流式细胞仪检测。
1)cDNA的合成及荧光定量PCR的检测
Trizol法提取细胞的总RNA，并按逆转录试剂盒的操作说明反转总RNA合成cDNA。然后用合成的cDNA按Promega公司的使用说明进行IL17A、IL17F基因的荧光定量PCR检测(以GAPDH基因为内参)，结果如图2所示。
如图2所示，体外Th17细胞分化实验验证α-倒捻子素能够抑制RORγt的靶基因IL17A分子的转录表达。
2)流式细胞仪检测
用IL17A和CD4的抗体进行细胞内染色，ELISA试剂盒检测冻存的细胞上清中IL17A的含量。实验操作严格按照eBioscience细胞内染色试剂盒和流式细胞仪操作说明进行，结果如图3所示。
如图3所示，细胞内的IL17A表达受到α-倒捻子素明显的抑制。
最后所应当说明的是，以上实施例用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者同等替换，而不脱离本发明技术方案的实质和范围。
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Claims (8)
1.α-倒捻子素在制备用于治疗自身免疫疾病的药物中的应用，所述α-倒捻子素的结构式为：
2.如权利要求1所述的应用，其特征在于：所述自身免疫疾病为Th17细胞介导的自身免疫疾病。
3.如权利要求1所述的应用，其特征在于：所述自身免疫疾病包括牛皮癣、多发性硬发症、风湿性关节炎、系统性红斑狼疮、特征性皮炎、哮喘和炎症性肠道病。
4.一种药物组合物，其特征在于：所述药物组合物包括α-倒捻子素和药学上可接受的载体，所述α-倒捻子素的结构式为：
5.如权利要求4所述的药物组合物，其特征在于：所述药物组合物的剂型是注射液或冻干制剂。
6.如权利要求4或5所述的药物组合物在制备用于治疗自身免疫疾病的药物中的应用。
7.如权利要求6所述的应用，其特征在于：所述自身免疫疾病为Th17细胞介导的自身免疫疾病。
8.如权利要求6所述的应用，其特征在于：所述自身免疫疾病包括牛皮癣、多发性硬发症、风湿性关节炎、系统性红斑狼疮、特征性皮炎、哮喘和炎症性肠道病。
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