CN1821423B - 一种抗关节炎药物细胞靶标筛选系统及筛药的方法 - Google Patents
一种抗关节炎药物细胞靶标筛选系统及筛药的方法 Download PDFInfo
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Abstract
本发明建立了一种抗关节炎药物的细胞靶标筛选系统及筛药的方法。本发明建立的细胞靶标筛药系统是运用分子克隆技术，分别将mPGES-1 promoter、COX-1 promoter和COX-2promoter部分基因克隆进人细胞株，获得在基因水平上表达的筛药细胞靶标M1、M2和M3，进而共同组成细胞靶标筛药系统。通过测定被筛选化合物抑制M1、M2和M3细胞luciferase表达的程度来筛选抗关节炎药物。
Description
技术领域
本发明涉及到一种抗关节炎药物筛选的新方法，更具体地说涉及到化合物对酶mPGES-1promoter表达的特异性抑制来评价该化合物是否有希望发展成为新型抗关节炎药物。
背景技术
关节炎是一种常见的慢性累及关节的炎症性疾病，即病变损害的部位是人体的关节，关节有红、肿、热、痛，功能障碍的炎症表现，或病理上构成关节的各组织有变质、渗出、增生性改变。最常见的是骨关节炎和类风湿性关节炎两种，我国目前关节炎患者估计有1亿以上，且人数还在不断增加。
关节炎患者炎症发生时，细胞膜上的磷脂在磷脂酶A2的催化下释放花生四烯酸(AA)，AA随之在环氧化酶(COX)作用下生成PGG2/PGH2，PGH2在不同酶的催化下产生前列腺素类物质和血栓烷(TXA2)，如在前列腺素E合成酶(PGES)作用下生成PGE2。其中，PGE2是主要的炎症介质，参与炎症、发热、疼痛反应，参与骨吸收和癌症；而PGD2、PGF2α为抗炎性前列腺素。TXA2是由血小板产生，有使血小板聚集和血管收缩的作用。PGI2在血管内皮细胞组成性表达，PGI2/TXA2的平衡对维持心血管安全方面起着重要作用。环氧化酶(COX)催化AA生成PGG2/PGH2，主要有两种：COX-1和COX-2。COX-1是组成性酶，结构性表达于几乎所有组织中，维持胃、肾、血小板等组织器官的生理功能。COX-2为诱导酶，炎症反应时受炎症因子刺激在炎症组织大量表达，促进PGE2的大量生成而参与炎症反应。对COX的认识产生目前治疗RA的一大类药物：非甾体抗炎免疫药(NSAIDs)。以对COX的抑制活性分为三类，非特异性抑制COX活性的NSAID，如阿司匹林、吲哚美辛、奇诺力、氟灭酸、扶他林、布洛芬、芬必得等；倾向性COX-2抑制剂，如尼美舒利、美罗昔康、氯诺昔康；特异性COX-2抑制剂，如Celecoxib(塞来昔布)、Rofecoxib(罗非昔布)、Valdecoxib(伐地昔布)，特异性COX-2抑制剂减轻了COX-1被抑制引起的胃肠道等不良反应，但由于COX-2在内皮细胞、前列腺、肺等器官以及正常肾脏皮质的致密斑及髓质间质细胞中也有表达，特异性COX-2抑制剂有延缓溃疡面的愈合，引起肾脏的不良反应，增加血栓形成的危险，增加气喘病人支气管哮喘的可能性。最近，Rofecoxib(罗非昔布)、Valdecoxib(伐地昔布)即因为增加病人心血管发病的几率而相继被厂家和FDA召回，同时FDA发布所有非甾体抗炎免疫药的最新用药指导，就如何使用NSAIDs发表了声明，综合现有数据，所有NSAIDs均有潜在的心血管危险。
前列腺素E合成酶(PGES)是PGE2生物合成过程的末端限速酶，催化环氧合酶的产物PGH2转化成PGE2，根据PGES在细胞中的定位和对谷胱甘肽的依赖性，可将PGES分为三种：cPGES、mPGES-1和mPGES-2。(1)胞浆PGES(cPGES)，为谷胱甘肽依赖型的组成性酶，表达在多种组织和细胞中不受炎症刺激因子的影响。cPGES的氨基酸序列与人的p23蛋白部分序列一致，是一23KD的伴侣分子，细胞中其活性的调节依赖于与hsp90的结合，催化COX-1来源的PGH2转化为PGE2；(2)mPGES-1：为一16KD的膜结合蛋白，与微粒体GSH-S-转移酶(MGST1)在氨基酸序列上有38％的同源性，以前命名为PIG12和MGST1-L1。广泛分布于各种组织和细胞中，是GSH依赖型的诱导型酶。在体外可被诱导COX-2的促炎症反应因子所诱导，包括LPS、IL-1β和TNFα等，其高水平表达于肺癌A549、HeLa、结肠癌、子宫腺癌和动脉粥样硬化斑块中，中度表达在胎盘、前列腺、睾丸、乳腺、膀胱和肾脏中。IL-1β诱导人肺癌A549细胞、IL-1β或TNFα诱导人类风湿性关节炎滑膜细胞、TNFα或LPS诱导鼠腹膜巨噬细胞共同表达mPGES-1和COX-2，mPGES-1与诱导型的COX-2相偶联，促进后续PGE2的合成。类风湿性关节炎患者中，mPGES-1主要于活动期滑膜细胞，而在静止期其表达几乎不能检出；另一方面，mPGES-2组成性表达，在活动期和静止期均能检出，存在于滑膜细胞和滑膜组织。同mPGES-1+/+小鼠相比，mPGES-1-/-小鼠具有明显降低炎症效应的作用。缺失mPGES-1的鼠腹腔细胞被LPS刺激时，失去产生PGE2的能力。在关节炎动物模型AIA模型中，对mPGES-1、mPGES-2、cPGES/p23、PGI2、COX-1、COX-2表达的mRNA和蛋白水平分析表明，mPGES-1表达可增加50-80倍，类似于COX-2。mPGES-2在第1天到15天间下降2-3倍，cPGES和COX-1能被诱导，但水平远低于mPGES-1(仅2-6倍)。PGI2在第1天短暂升高，3天后到第25天之间PGIS mRNA表达下降3-9倍，说明其在AIA慢性炎症维持中发挥愈小的作用。(3)mPGES-2：与mPGES-1不同，其是非GSH依赖型、组成型表达的酶，不被组织炎症和损伤显著诱导，但在人结肠癌中，表达有显著增加。主要存在于大脑、心脏、脾脏、子宫、肾、肝及骨骼肌肉中，mPGES-2作为一高尔基体膜相关蛋白被合成，自动切割其N端疏水区成为成熟蛋白分布在胞浆中，并有在核周边聚集的趋势。可通过COX-1和COX-2介导PGE2的产生，在组织体内平衡和疾病中发挥作用。
因此开发选择性抑制mPGES-1表达或酶活性从而抑制PGE2生成的药物将具有重大的社会意义和经济价值。为了避免目前环氧化酶(COX-1、COX-2)抑制剂药物的副作用，我们以COX-1和COX-2作为对照筛选的靶标。
发明内容
本发明的目的是提供一种抗关节炎药物的细胞靶标筛选系统。
本发明的另一目的是提供应用细胞靶标筛选系统筛选抗关节炎药物的方法。
本发明建立了一种抗关节炎药物的细胞靶标筛选系统及筛药方法。本发明建立的细胞靶标筛药系统是运用分子克隆技术，分别将mPGES-1 promoter、COX-1 promoter和COX-2promoter基因克隆进pGL3BN载体Luciferase基因上游的克隆位点Xho I/Hind III、NheI/Xho I、Xho I/Bgl II之间，从而构建重组载体pGL3BN/mPGES-1 promoter、pGL3BN/COX-1promoter、pGL3BN/COX-2 promoter，然后将重组质粒pGL3BN/mPGES-1 promoter、pGL3BN/COX-1 promoter、pGL3BN/COX-2 promoter利用脂质体转染法分别转染人A549细胞株，运用细胞克隆的有限稀释法筛选得到基因水平上稳定表达的筛药细胞靶标M1、M2和M3(mPGES-1 promoter的表达会启动其下游Luciferase的表达，也就是说测定Luciferase表达的水平可以反映mPGES-1 promoter表达的程度，同理也适用于COX-1 promoter和COX-2promoter)，进而共同组成细胞靶标筛药系统。
如上所述利用脂质体转染法获取稳定转染细胞株的详细操作为：将10ul脂质体加入100ul不含血清和抗生素的RPMI 1640培养液混匀，共做三份(溶液A)；然后各取2ugpGL3BN/mPGES-1 promoter、pGL3BN/COX-1 promoter、pGL3BN/COX-2 promoter质粒DNA分别加入100ul不含血清和抗生素的RPMI 1640培养液混匀(溶液B、C、D)。分别将溶液B、C、D与一份溶液A混合，室温搁置40分钟，备用。转染前一天，A549细胞以3×105数进6孔培养板中的每一孔，加入1ml含10％胎牛血清的RPMI 1640培养液。转染时，用2ml RPMI 1640培养液洗细胞一次，加入0.8ml RPMI 1640培养液入混合液，将三份混合液分别加于三孔细胞上，细胞与质粒在培养箱中于5％ CO2和37℃条件下转染24小时。将培养液换成正常培养液，即含10％胎牛血清的RPMI 1640培养液，培养48小时，形成瞬转染细胞。将瞬转染细胞以2000细胞/100mm平皿的细胞分布密度分进培养皿，在含800ug/ml G418的RPMI 1640培养液中于37℃，5％ CO2培养箱中培养。三周后可见细胞集落形成，用10ul枪头挑取单个集落入48孔板扩增培养。待单克隆细胞长满培养板孔后，一部分细胞继续传代培养，一部分细胞测定luciferase表达，luciferase表达达1000/1×105cell以上且10代以上传代其表达无降低者，即为稳定转染细胞株。由此方法筛选得到稳定转染细胞株M1、M2和M3。
本发明所述的mPGES-1 promoter的基因序列为：
5’-cacagcttcattctctgacagttctggtggatgaagcctgaaatgggtctcacagggctaaaatcaaagtgtcgggagggctgcgttctgtctgaacgcagagagtttgtttcgtccccttttccagtttctagaggcccctggtattccttgggccatgatctctccttcatcttcaaggccagcaggttagcgtctctcctgtctgacccctattctgtcctttcatctcctctcttaatgctgcctctgtttttatatcttttctctaattttactgcctccctcttggaagggcccctctgtatactctgggctcacccagataaactaggataatttctcccatctcaaatccttacgaaatcccatgtgaaaagtcccttttgccacatagtcacagtcacgggttctagggattgggacatggatgtctttaggggtggagggcgtccttcagcctgccacagatggcaaagtcatgccattgtggctgcagctccattgtccaggctgagtgtgggggcgcatggcgtggttctcatgcccaccacctaccctgaccctgcggaggtgggcaggaggcagcgataatcaccacgtgcagaggtcctgccaggtgcccagcctcatcattccctagaaggcaggcaccaccacatgctcccacactgtagataaggaaacagctttgaaacagatttgaaagtgtttgaggatttgcctggaaggagggctccagagccagggactgggtccgggacacccggagactctctgcttcttggagctccggcaactgcttgtctttctcttcacaggagaagggggccctgctgccgctgccgtggggcggggcgtgggcggtgctggctgcaggaaggctctcccctcccactttgtgctttccctgcaggctgctcctctgtcgagctgatcacacccacagttgagct-3’
本发明所述的COX-1 promoter的基因序列为：
5’-atgggaacaccttgggtgagaatgggggaggggcttcccgcaggtttcacctctcccacctggcataatttctaccctgcacccagtgatgcccaggagccagcacaagaacctctgagctctcctgccggctttccccactgtggggcccacaggatggctgggtgtgctggggaaactgagggagatgacttgtgggtgccagcaccatgggactttgggtgatcttggacctattccttcctccttctaggtgcttcctgagctggcgaaaaggagagctggatgctgggattctataaattcggcggtggatgtgagtctagctacagcatggaccctggccagccctggaatctgagttcagagatctttgaaaaaatgccttccgataactgagcacctactacatctggacactgcaccaggagatttgtgtgcattccctcattgaatcttacaccaccctctgggatggtttttgctattaagccgattttatagatgagaatactgagggctagaaaagataagtaccttgtccaaggtgacacggccagtaagctgtggagtctggatttgaactaggctctcttggagtttggagcccggtcttttcatttctggcagagacctgtggcttcagggggcggagccagccccatgggggaagtaagtccttcctgttagatctgtctgaggagtagtgagtttctcatctaggaaaccatcagcaggccaggctggactgccccatttctagccccctttcttctctgcagactgccctgtttctagccccttttcttctctacccttttctgtagagtgcgttagacccattttacagaaggggactctgagaccctttcttctctgcggggcagggtatgtggttcagacgagccgcttccatccccagttctgtccctaagccctgggtgacctcagcattgagcatcgtctctgagcctcagtttcctgtctatatctagtgccaccaggcatcagaaacgtaagtgcttcaaggatcttgggtgaaaagccgctttagcggcgagcatacactaattaattaaaatgccttggccggggctgggcagaggaagtaagcgggcagccgaggtgacagctggagggaggagcgggggtggagccgggggaagggtggggaggggatgggctggagctccgggcagtgtgcgaggcgcacgcacaggagcctgcactctgcgtcccgcaccccagcagccgcg-3’
本发明所述的COX-2 promoter的基因序列为：
5’-attattaaattatcaaaaagaaaatgatccacgctcttagttgaaatttcatgtaagattccatgcaataaataggagtgccataaatggaatgatgaaatatgactagaggaggagaaaggcttcctagatgagatggaattttagtcatccgtgtctcatgaagaatcagatgtgtacactaagcaaaacagttaaaaaaaaaacctccaagtgagtctcttatttatttttttcttataagacttctacaaattgaggtacctggtgtagttttatttcaggttttatgctgtcattttcctgtaatgctaaggacttaggacataactgaattttctattttccacttcttttctggtgtgtgtgtatatatatatgtatatatacacacacacatatacatatatatattttttagtatctcaccctcacatgctcctccctgagcactacccatgatagatgttaaacaaaagcaaagatgaaattccagctgtcaaaatctcccttccatctaattaattcctcatccaactatgttccaaaacgagaatagaaaattagccccaataagcccaggcaactgaaaagtaaatgctatgttgtactttgatccatggtcacaactcataatcttggaaaagtggacagaaaagacaaaagagtgaactttaaaactcgaatttattttaccagtatctcctatgaagggctagtaaccaaaataatccacgcatcagggagagaaatgccttaaggcatacgttttggacatttagcgtccctgcaaattctggccatcgccgcttcctttgtccatcagaaggcaggaaactttatattggtgacccgtggagctcacattaactatttacagggtaactgcttaggaccagtattatgaggagaatttacctttcccgcctctctttccaagaaacaaggagggggtgaaggtacggagaacagtatttcttctgttgaaagcaacttagctacaaagataaattacagctatgtacactgaaggtagctatttcattccacaaaataagagttttttaaaaagctatgtatgtatgtgctgcatatagagcagatatacagcctattaagcgtcgtcactaaaacataaaacatgtcagcctttcttaaccttactcgccccagtctgtcccgacgtgacttcctcgaccctctaaagacgtacagaccagacacggcggcggcggcgggagaggggattccctgcgcccccggacctcagggccgctcagattcctggagaggaagccaagtgtccttctgccctcccccggtatcccatccaaggcgatcagtccagaactggctctcggaagcgctcgggcaaagactgcgaagaagaaaagacatctggcggaaacctgtgcgcctggggcggtggaactcggggaggagagggagggatcagacaggagagtggggactaccccctctgctcccaaattggggcagcttcctgggtttccgattttctcatttccgtgggtaaaaaaccctgcccccaccgggcttacgcaatttttttaaggggagaggagggaaaaatttgtggggggtacgaaaaggcggaaagaaacagtcatttcgtcacatgggcttggttttcagtcttataaaaaggaaggttctctcggttagcgaccaattgtcatacgacttgcagtgagcgtcaggagcacgtccaggaactcctcagcagcgcctccttcagctccacagccagacgccctcagacagcaaagcctacccccgcgccgcgccctgcccgccgctgcgatgctcgcccgcgccctgctgctgtgcgcggtcctggcg-3’
被筛选化合物对mPGES-1 promoter表达的影响可通过其下游luciferase报告基因的表达水平来说明，也就是说被筛选化合物和M1细胞共同培养后通过测定M1细胞luciferase基因表达水平可反映被筛选化合物对mPGES-1 promoter表达的影响。同理通过测定M2、M3细胞luciferase基因表达水平可分别反映被筛选化合物对COX-1 promoter和COX-2promoter表达的影响。
通过测定被筛选化合物作用于M1细胞luciferase基因表达的降低来筛选抑制mPGES-1promoter表达的物质；通过测定被筛选化合物作用于M2细胞luciferase基因表达的降低来筛选抑制COX-1 promoter表达的物质；通过测定被筛选化合物作用于M3细胞luciferase基因表达的降低来筛选抑制COX-2 promoter表达的物质。我们定义mPGES-1 promoter特异性抑制剂为对mPGES-1 promoter的抑制率大于40％而对COX-1 promoter和COX-2 promoter的抑制率均小于10％。通过筛选mPGES-1 promoter特异性抑制剂来筛选抗关节炎药物。
本发明的另一目的提供了应用上述细胞靶标筛药系统筛选抗关节炎药物的方法，该方法包括以下步骤：
(1)用稳定转染细胞株M1、M2和M3，分别以5*104个/孔细胞接种于48孔培养板中，37℃培养一天，吸去昨日的旧培养液，换上新培养液，体积200ul/孔。
(2)加被筛选物质(不同来源的化合物或混合物)于细胞培养液内和细胞培养一天。
(3)吸去细胞培养液，用0.9％NaCl洗2遍。
(4)加入1×CCLR 100ul/孔覆盖细胞。
(5)充分吹打细胞，将细胞碎片和液体转入离心管，12,000×g离心5秒，将上清转入新管中。
(6)将20ul细胞抽提液与100ul的Luciferase Assay Reagent(室温平衡)用枪头混匀。
(7)将管子放入Luminometer中，按Go键开始检测。
(8)样品抑制率定义为(1-样品的荧光值/对照的荧光值)×100％，样品对M1细胞的抑制率大于40％且同时对M2和M3的抑制率小于10％为中选化合物。
本发明的细胞靶标系统可用于筛选合成的单个或复合化合物，筛选植物提取的单个化合物或混合物，筛选各种微生物代谢产物中选择性抑制mPGES-1的物质从而达到抑制PGE2生成的目的。
附图说明
图1、建立由mPGES-1 promoter、COX-1 promoter和COX-2 promoter基因分别稳定转染的细胞株组成的筛药系统及测定Luciferase路线图。
运用分子克隆技术，分别将mPGES-1 promoter、COX-1 promoter和COX-2 promoter基因部分克隆进pGL3BN载体Luciferase基因上游的克隆位点Xho I/Hind III、Nhe I/Xho I、Xho I/Bgl II之间，从而构建重组载体pGL3BN/mPGES-1 promoter、pGL3BN/COX-1 promoter、pGL3BN/COX-2 promoter，然后将重组载体pGL3BN/mPGES-1 promoter、pGL3BN/COX-1promoter、pGL3BN/COX-2 promoter利用脂质体转染法分别转染人A549细胞株，运用细胞克隆的有限稀释法筛选得到基因水平上稳定表达的筛药细胞靶标M1、M2和M3。
图2、利用所建细胞靶标筛药系统对化合物CM188的筛选。
具体实施方式
实施例
(1)稳定转染细胞株M1、M2和M3分别以5*104个/孔细胞接种于48孔培养板中，37℃培养一天，吸去昨日的旧培养液，换上新培养液，体积200ul/孔。
(2)分别加终浓度为0ug/ml、10ug/ml、30ug/ml、100ug/ml、300ug/ml的吲哚美辛、化合物CM188、CM123、槲皮素、次野尾素于细胞培养液内和细胞培养一天。吸去细胞培养液，用0.9％NaCl洗2遍。
(3)加入1×CCLR 100ul/孔覆盖细胞。充分吹打细胞，将细胞碎片和液体转入离心管，12,000×g离心5秒，将上清转入新管中。
(4)将20ul细胞抽提液与100ul的Luciferase Assay Reagent(室温平衡)用枪头混匀。将管子放入Luminometer中，按Go键开始检测。
结果如下表所示，其中吲哚美辛、槲皮素、次野尾素对细胞M1的mPGES-1 promoter表达的抑制率均大于40％，但是吲哚美辛、次野尾素对M2细胞COX-1 promoter、M3细胞COX-2 promoter表达的抑制率也均大于10％；槲皮素虽然对M3细胞无抑制，但其对M2细胞抑制率远大于10％，故吲哚美辛、槲皮素、次野尾素均不符合中选化合物的定义，予以淘汰。而化合物CM188、CM123对细胞M1的mPGES-1 promoter表达的抑制率可达到68.16％和45.81％，对M2细胞COX-1 promoter表达的抑制率在筛药浓度范围内最大为9.31％和3.55％，小于10％，对M3细胞COX-2 promoter的表达抑制率为8.67％或无抑制，符合我们对中选化合物的定义，故化合物CM188、CM123是特异性抑制mPGES-1 promoter表达的化合物，对其进行进一步的药理药效学的评价可有望发展成新的抗关节炎药物。
注：“-”表示无抑制作用，“×”表示不符合中选化合物的定义，“√”表示符合中选化合物的定义，可进一步开发。
Claims (4)
1.一种抗关节炎药物的细胞靶标筛选细胞株，由筛药细胞株M1、M2和M3组成，它们分别由如下步骤制备而得：
a)分别将mPGES-1 promoter、COX-1 promoter和COX-2 promoter基因克隆进pGL3BN载体Luciferase基因上游的克隆位点Xh0 I/Hind III、Nhe I/Xho I、Xho I/BglII之间，从而获得重组载体pGL3BN/mPGES-1 promoter、pGL3BN/COX-2 promoter、pGL3BN/COX-2 promoter，
所述的mPGES-1 promoter、COX-1 promoter和COX-2 promoter基因序列分别为：
mPGES-1 promoter基因序列为：
5’-cacagcttcattctctgacagttctggtggatgaagcctgaaatgggtctcacagggctaaaatcaaagtgtcgggagggctgcgttctgtctgaacgcagagagtttgtttcgtccccttttccagtttctagaggcccctggtattccttgggccatgatctctccttcatcttcaaggccagcaggttagcgtctctcctgtctgacccctattctgtcctttcatctcctctcttaatgctgcctctgtttttatatcttttctctaattttactgcctccctcttggaagggcccctctgtatactctgggctcacccagataaactaggataatttctcccatctcaaatccttacgaaatcccatgtgaaaagtcccttttgccacatagtcacagtcacgggttctagggattgggacatggatgtctttaggggtggagggcgtccttcagcctgccacagatggcaaagtcatgccattgtggctgcagctccattgtccaggctgagtgtgggggcgcatggcgtggttctcatgcccaccacctaccctgaccctgcggaggtgggcaggaggcagcgataatcaccacgtgcagaggtcctgccaggtgcccagcctcatcattccctagaaggcaggcaccaccacatgctcccacactgtagataaggaaacagctttgaaacagatttgaaagtgtttgaggatttgcctggaaggagggctccagagccagggactgggtccgggacacccggagactctctgcttcttggagctccggcaactgcttgtctttctcttcacaggagaagggggccctgctgccgctgccgtggggcggggcgtgggcggtgctggctgcaggaaggctctcccctcccactttgtgctttccctgcaggctgctcctctgtcgagctgatcacacccacagttgagct-3’，
COX-1 promoter的基因序列为：
5’-atgggaacaccttgggtgagaatgggggaggggcttcccgcaggtttcacctctcccacctggcataatttctaccctgcacccagtgatgcccaggagccagcacaagaacctctgagctctcctgccggctttccccactgtggggcccacaggatggctgggtgtgctggggaaactgagggagatgacttgtgggtgccagcaccatgggactttgggtgatcttggacctattccttcctccttctaggtgcttcctgagctggcgaaaaggagagctggatgctgggattctataaattcggcggtggatgtgagtctagctacagcatggaccctggccagccctggaatctgagttcagagatctttgaaaaaatgccttccgataactgagcacctactacatctggacactgcaccaggagatttgtgtgcattccctcattgaatcttacaccaccctctgggatggtttttgctattaagccgattttatagatgagaatactgagggctagaaaagataagtaccttgtccaaggtgacacggccagtaagctgtggagtctggatttgaactaggctctcttggagtttggagcccggtcttttcatttctggcagagacctgtggcttcagggggcggagccagccccatgggggaagtaagtccttcctgttagatctgtctgaggagtagtgagtttctcatctaggaaaccatcagcaggccaggctggactgccccatttctagccccctttcttctctgcagactgccctgtttctagccccttttcttctctacccttttctgtagagtgcgttagacccattttacagaaggggactctgagaccctttcttctctgcggggcagggtatgtggttcagacgagccgcttccatccccagttctgtccctaagccctgggtgacctcagcattgagcatcgtctctgagcctcagtttcctgtctatatctagtgccaccaggcatcagaaacgtaagtgcttcaaggatcttgggtgaaaagccgctttagcggcgagcatacactaattaattaaaatgccttggccggggctgggcagaggaagtaagcgggcagccgaggtgacagctggagggaggagcgggggtggagccgggggaagggtggggaggggatgggctggagctccgggcagtgtgcgaggcgcacgcacaggagcctgcactctgcgtcccgcaccccagcagccgcg-3’，
COX-2 promoter的基因序列为：
5’-attattaaattatcaaaaagaaaatgatccacgctcttagttgaaatttcatgtaagattccatgcaataaataggagtgccataaatggaatgatgaaatatgactagaggaggagaaaggcttcctagatgagatggaattttagtcatccgtgtctcatgaagaatcagatgtgtacactaagcaaaacagttaaaaaaaaaacctccaagtgagtctcttatttatttttttcttataagacttctacaaattgaggtacctggtgtagttttatttcaggttttatgctgtcattttcctgtaatgctaaggacttaggacataactgaattttctattttccacttcttttctggtgtgtgtgtatatatatatgtatatatacacacacacatatacatatatatattttttagtatctcaccctcacatgctcctccctgagcactacccatgatagatgttaaacaaaagcaaagatgaaattccagctgtcaaaatctcccttccatctaattaattcctcatccaactatgttccaaaacgagaatagaaaattagccccaataagcccaggcaactgaaaagtaaatgctatgttgtactttgatccatggtcacaactcataatcttggaaaagtggacagaaaagacaaaagagtgaactttaaaactcgaatttattttaccagtatctcctatgaagggctagtaaccaaaataatccacgcatcagggagagaaatgccttaaggcatacgttttggacatttagcgtccctgcaaattctggccatcgccgcttcctttgtccatcagaaggcaggaaactttatattggtgacccgtggagctcacattaactatttacagggtaactgcttaggaccagtattatgaggagaatttacctttcccgcctctctttccaagaaacaaggagggggtgaaggtacggagaacagtatttcttctgttgaaagcaacttagctacaaagataaattacagctatgtacactgaaggtagctatttcattccacaaaataagagttttttaaaaagctatgtatgtatgtgctgcatatagagcagatatacagcctattaagcgtcgtcactaaaacataaaacatgtcagcctttcttaaccttactcgccccagtctgtcccgacgtgacttcctcgaccctctaaagacgtacagaccagacacggcggcggcggcgggagaggggattccctgcgcccccggacctcagggccgctcagattcctggagaggaagccaagtgtccttctgccctcccccggtatcccatccaaggcgatcagtccagaactggctctcggaagcgctcgggcaaagactgcgaagaagaaaagacatctggcggaaacctgtgcgcctggggcggtggaactcggggaggagagggagggatcagacaggagagtggggactaccccctctgctcccaaattggggcagcttcctgggtttccgattttctcatttccgtgggtaaaaaaccctgcccccaccgggcttacgcaatttttttaaggggagaggagggaaaaatttgtggggggtacgaaaaggcggaaagaaacagtcatttcgtcacatgggcttggttttcagtcttataaaaaggaaggttctctcggttagcgaccaattgtcatacgacttgcagtgagcgtcaggagcacgtccaggaactcctcagcagcgcctccttcagctccacagccagacgccctcagacagcaaagcctacccccgcgccgcgccctgcccgccgctgcgatgctcgcccgcgccctgctgctgtgcgcggtcctggcg-3’；
b)将构建好的重组质粒pGL3BN/mPGES-1 promoter、pGL3BN/COX-1 promoter、pGL3BN/COX-2 promoter利用脂质体转染法分别转染人A549细胞株；
c)分别利用有限稀释法筛选得到基因水平上稳定表达的筛药细胞株M1、M2和M3。
2.根据权利要求1所述的抗关节炎药物的细胞靶标筛选细胞株，其特征在于转染人A549细胞通过将脂质体加入不含有血清和抗菌素的RPMI1640培养液中混匀获得溶液A，分做三份，然后将pGL3BN/mPGES-1 promoter、pGL3BN/COX-1 promoter、pGL3BN/COX-2promoter质粒DNA分别与上述的不含血清和抗生素的RPMI1640培养液混匀获得溶液B、C、D，分别将溶液B、C、D与一份溶液A混合备用，然后与A549细胞转染形成瞬转染细胞，然后在含10％胎牛血清的RPMI1640培养液中于37℃5％CO2培养箱培养，测定Luciferase表达即得稳定转染细胞株；用此方法筛选得稳定转染细胞株M1、M2、M3。
3.根据权利要求1所述的抗关节炎药物的细胞靶标筛选细胞株，其特征在于被筛选化合物通过mPGES-1 promoter Luciferase报告基因的表达水平反映被筛选化合物对mPGES-1 promoter表达的影响；通过测定M2、M3细胞Luciferase基因表达水平分别反映被筛化合物对COX-1 promoter或COX-2 promoter表达影响。
4.根据权利要求1所述的抗关节炎药物的细胞靶标筛选细胞株的筛选抗关节炎药物的方法，包括如下步骤：
1)培养M1、M2、M3稳定转染细胞株得培养液；
2)加入被筛选化合物到上述培养液中，和细胞培养一天；
3)吸去细胞培养液，用稀NaCl水洗，加入1×CCLR 100ul/孔覆盖细胞；
4)充分吹打细胞，将细胞碎片和液体离心，上清转入新管中；
5)将细胞抽提液与Luciferase试剂用枪头混匀；
6)将管子放入Luminometer中检测样品抑制率。
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