CN103983770A - 一种小分子抗炎药物的筛选方法 - Google Patents

一种小分子抗炎药物的筛选方法 Download PDF

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CN103983770A
CN103983770A CN201410196240.3A CN201410196240A CN103983770A CN 103983770 A CN103983770 A CN 103983770A CN 201410196240 A CN201410196240 A CN 201410196240A CN 103983770 A CN103983770 A CN 103983770A
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孙兵
侯建
马丽艳
伊春艳
王江君
凌志洋
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Institut Pasteur of Shanghai of CAS
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Abstract

本发明提供一种高通量筛选小分子抗炎药物的方法，利用LPS（内毒素）刺激腹腔巨噬细胞来模拟体内炎症发生时巨噬细胞被激活这一生物学现象。通过检测药物对巨噬细胞分泌细胞因子的影响作用来评价药物的抗炎活性。

Description

一种小分子抗炎药物的筛选方法
技术领域
本发明涉及到一种抗炎药物筛选的新方法，更具体地说是从细胞水平角度，通过化合物对腹腔巨噬细胞分泌细胞因子的影响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
炎性反应，俗称发炎，指生物组织受到外伤、出血、或病原感染等刺激，激发的生理反应，是先天免疫系统为移除有害刺激或病源体及促进修复的保护措施，其中包括了红肿、发热、疼痛等症状。通常情况下，炎症是有益的，是人体的自动的防御反应，但是有的时候，炎症可以引起人体自身免疫系统的过敏，进而攻击自身的组织及细胞、如类风湿性关节炎和红班狼疮症等免疫系统过敏病症，免疫系统过敏所生成的COX-2及IL-1β，使到软骨组织疼痛及发炎。长期发炎可引起一系列疾病，如花粉症，牙周炎，动脉粥样硬化，类风湿性关节炎，甚至癌症（如胆囊癌）。炎性反应可分为急性炎症和慢性炎症。急性炎症是生物体应该有害刺激的初步反应，更多的血浆和白血球(特别是粒细胞)从血液移往受损组织。一连串的生化反应进行传播并熟成进一步的炎性反应，当中牵涉局部的血管系统，免疫系统及受损组织内的各个细胞。慢性炎症引致发炎部位的细胞类型改变，组织的毁灭与修复同时进行。
抗炎药物目前主要有两类，一类是NSAIDs（非甾体抗炎药），是一类疾病症状改善类药物，以COX酶抑制剂居多。它通过抑制前列腺素的合成而缓解炎症的红肿热痛等症状，但是并无法阻止慢性炎症的发展进程，对疾病的发展没有很好的控制作用。而且这一类药物以COX酶为靶标，存在潜在的心脑血管和胃肠道出血的风险。另一类则是一些免疫调剂药物，这一类药物可以通过干预异常免疫应答的进程，从而可以有效控制慢性炎症，如自身免疫病的疾病的发展。
炎症是一个复杂的过程，炎症发生过程中涉及到多种细胞的活化和细胞因子的 释放。其中，巨噬细胞是参加炎症反应的一种重要的细胞。巨噬细胞是IL-l β、TNF-a和IL-6等细胞因子的主要来源，针对这几个炎性因子的生物大分子在临床应用上也取得了成功，于是我们选择巨噬细胞作为我们的筛选体系，选择IL-l β、TNF-a和IL-6作为我们的评价标准。
目前，科研人员主要利用永生化的人或啮齿类动物的细胞系进行抗炎药物的筛选。这种筛选方式能够实现药物的快速筛选，但由于体外培养的永生化细胞已失去体内细胞的一些活性和特征，筛选结果不能完全反映药物在体内的作用情况。而原代培养的细胞与生物体内细胞性状相似性大，更能反映细胞在体内真实的功能状态。因此将原代培养的细胞模型应用到抗炎药物的筛选将会弥补当前筛药方法的不足。
发明内容
本发明的目的是提供一种抗炎药物的细胞靶标筛选系统。从6-8周雌性Balb/c小鼠腹腔中分离巨噬细胞，在96孔板培养后用内毒素（LPS）进行处理，激活巨噬细胞。与此同时采用待测药物处理，用ELISA检测细胞培养上清中炎症细胞因子TNF-α，IL-1β以及IL-6的表达水平，评价药物的抗炎效果。
附图说明
图1：利用所建筛选系统TNF-α指标对阴性阳性对照药的药效评价。
图2：利用所建筛选系统IL-1β指标对阴性阳性对照药的药效评价。
图3：利用所建筛选系统IL-6指标对阴性阳性对照药的药效评价。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
1) 取8周雌性balb/c小鼠，腹腔注射4%的肉汤培养基；
2) 72-96小时后，处死小鼠，酒精浸泡5分钟；
3) 向小鼠腹腔内注射5mL 4℃预冷DMEM培液，腹部按摩5分钟；
4) 用10mL注射器吸取小鼠腹腔内的培液，转移到50mL离心管中；
5) 重复第三步和第四步一次；
6) 1500转/分钟离心3分钟，弃去上清，用37℃完全DMEM培养基重悬细胞；
7) 计数，调整细胞浓素至2×105/mL，铺在96孔板中培养，每孔100μL；
8) 2小时后，换培液，吸去尚未贴壁的细胞；
9) 培养24小时后，加入1μg/mL的LPS与特定浓度的待测小分子药物； 
10)   培养12小时后ELISA检测上清中细胞因子TNF-α和IL-6浓度（对于IL-1β的检测，在LPS刺激后需要去掉上清，并加入300μL含5mM ATP的无血清DMEM重刺激1h，再取上清检测）
结论
阳性对照药物SB202190（p38的抑制剂) 、YL064（本实验室开发的抗炎药物，已经证明有抗炎活性)均能在这个筛选系统中有效的抑制炎性细胞因子（TNF-α、IL-1β和IL-6）的产生，而阴性对照小分子药物YL064*（YL064衍生物，丧失了其抗炎作用）则无相应的抑制效果。结果重复性好，稳定，证明了该系统是一个可靠有效的抗炎药物筛选评价系统。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。

Claims (4)

1.一种小分子抗炎药物的筛选方法，其特征在于：所述的方法由如下步骤组成：a)从8周雌性Balb/c小鼠中分离出小鼠腹腔巨噬细胞，铺在96孔板中，并在37℃二氧化碳培养箱中培养；b)在铺好的细胞中加入指定浓度待测小分子药物共同孵育，与此同时加入内毒素进行刺激； c)药物、细胞和内毒素共同培养一段时间后，直接收集或者加入ATP处理后收集培养上清，检测炎症细胞因子的浓度。
2.根据权利要求1所述的筛选方法，其特征在于：采取小鼠腹腔原代巨噬细胞；通过向小鼠腹腔中注射不完全DMEM培养基，轻揉按摩后抽提出巨噬细胞，再将DMEM培养液取出从而获得原代巨噬细胞。
3.根据权利要求1所述的筛选方法，其特征在于：被筛选化合物通过TNF-α、IL-1β和IL-6的表达水平反映被筛选化合物的抗炎活性；通过测定培液上清中TNF-α、IL-1β和IL-6的浓度反映被筛化合物的抗炎与免疫调剂能力。
4.根据权利要求1所述的筛选方法，其特征在于：筛选抗炎药物的方法包括如下步骤：1)抽提培养原代的小鼠腹腔巨噬细胞于96孔板中；2)加入被筛选化合物到DMEM细胞培液中，再加入一定浓度的LPS，然后和细胞共培养一天；3)收集细胞培养液，得培养液1，PBS清洗细胞后，加入混有ATP的DMEM培养基；4)培养一个小时后收集培液2；5)用ELISA方法检测培养液1中的TNF-α、IL-6和培养液2中的IL-1β。
CN201410196240.3A 2014-05-12 2014-05-12 一种小分子抗炎药物的筛选方法 Pending CN103983770A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104833782B (zh) * 2015-05-08 2016-07-06 中国农业科学院兰州畜牧与兽药研究所 一种体外筛选和检测抗奶牛子宫内膜炎药物的方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104833782B (zh) * 2015-05-08 2016-07-06 中国农业科学院兰州畜牧与兽药研究所 一种体外筛选和检测抗奶牛子宫内膜炎药物的方法

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