CN111374989B - 用于治疗炎症性肠病的药物 - Google Patents

用于治疗炎症性肠病的药物 Download PDF

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CN111374989B
CN111374989B CN202010183029.3A CN202010183029A CN111374989B CN 111374989 B CN111374989 B CN 111374989B CN 202010183029 A CN202010183029 A CN 202010183029A CN 111374989 B CN111374989 B CN 111374989B
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inflammatory bowel
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CN111374989A (zh
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黄曦
王子晴
洪海
吴永坚
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Fifth Affiliated Hospital of Sun Yat Sen University
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Abstract

本发明属于免疫治疗技术领域,公开了一种用于治疗炎症性肠病的药物,所述药物包括CD4‑CD8‑双阴性的T细胞。通过构建炎症性肠病的小鼠模型,然后抽取自体外周血,分离出DN T细胞,体外扩增培养后,再静脉注射回输给自体,能够达到缓解治疗肠道炎症的目的。本发明首次利用DN T细胞及其免疫治疗方法可以实现对炎症性肠病的免疫治疗,其方法具有减轻肠道炎症等优点,适于炎症性肠病的综合治疗,适用于临床推广应用。

Description

用于治疗炎症性肠病的药物
技术领域
本发明具体涉及免疫治疗技术领域,更具体的,涉及用于治疗炎症性肠病的药物。
背景技术
炎症性肠病(inflammatory bowel disease,IBD)属于自身免疫性疾病,是一种发生在肠道的,要终生治疗并且不可治愈的慢性特发性疾病,具有频繁复发和缓解的临床特点。近十年来,IBD在我国就诊人数呈逐步增加趋势,将成为我国消化系统常见病。IBD的常规药物疗法,例如抗炎剂、糖皮质激素和免疫抑制剂由于其在长期治疗期间的全身不良反应和毒性而受到限制。
细胞治疗是通过体外激活、扩增自体或异体的免疫效应细胞,再回输给患者,起到杀伤目标细胞和提高患者免疫功能等作用。细胞免疫治疗是未来炎症性肠病综合治疗的重要组成部分。
现有技术还没有关于治疗炎症性肠病的细胞免疫治疗方法。
发明内容
针对上述现有技术存在的缺陷,本发明的主要目的是提供一种用于治疗炎症性肠病的药物。
本发明的目的通过以下技术方案实现:
一种用于治疗炎症性肠病的药物,所述药物包括CD4-CD8-双阴性的T细胞。
优选的,所述CD4-CD8-双阴性的T细胞为CD3+CD4-CD8-双阴性T细胞(简称为DN T细胞)。
本发明首先通过构建炎症性肠病的小鼠模型,然后抽取自体外周血,分离出DN T细胞,体外扩增培养后,再静脉注射回输给自体,能够达到缓解治疗肠道炎症的目的。
优选的,所述CD3+CD4-CD8-双阴性T细胞来自外周血。
优选的,所述外周血为自体外周血。当然,与自体血型匹配的受试者的外周血也是可以的。
优选的,所述自体外周血的CD3+CD4-CD8-双阴性T细胞通过流式分选方法获得,同时经过体外扩增培养。
更优选的,所述体外扩增培养过程中加入白介素-2,能够刺激体DN T细胞的扩增;所述白介素-2的浓度为20ng/mL。
与现有技术相比,本发明具有以下有益效果:
本发明通过构建炎症性肠病的小鼠模型,然后抽取自体外周血,分离出DN T细胞,体外扩增培养后,再静脉注射回输给自体,能够达到缓解治疗肠道炎症的目的。本发明首次利用DN T细胞及其免疫治疗方法可以实现对炎症性肠病的免疫治疗,其方法具有减轻肠道炎症等优点,适于炎症性肠病的综合治疗,适用于临床推广应用。
附图说明
图1为输入DN T细胞受体肠道病理切片;
图2为输入DN T细胞受体小鼠体重下降情况,其中DN T组即为回输组;
图3为输入受体小鼠的供体DN T细胞分泌情况;其中,PMA+Ion+BFA组代表小鼠DNT细胞体外刺激组,PMA为佛波酯,PMA和Ionomycin的共同作用可以起到激活细胞的作用。
BFA为蛋白转运抑制剂,阻止高尔基体将新分泌蛋白运输到细胞外,从而达到通过流式来检测细胞因子的目的;unstimulated代表未加刺激组。
具体实施方式
为了对本发明的技术特征、目的和效果有更加清楚的理解,现对照附图详细说明本发明的具体实施方式。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1 以小鼠为模型建立用于免疫治疗炎症性肠病的DN T细胞的方法
一、IBD小鼠模型的建立过程
使用葡聚糖硫酸钠(dextran sulfate sodium,DSS)诱导小鼠结肠炎模型,给小鼠饮用含3%(w/v)DSS水持续7天,再改用正常水饮用1天,构建小鼠结肠炎模型。每日检测小鼠体重及疾病活动度指数(DAI),用来评价小鼠结肠炎严重程度。DAI评分包括大便性状,直肠出血情况等。
二、向建立的小鼠模型中回输DN T细胞
用于免疫治疗炎症性肠病的DN T细胞的方法用到以下成分:(1)T细胞的特异性荧光抗体;(2)培养基(10%胎牛血清的1640培养基);(3)白介素-2(20ng/ml)。
具体包括如下步骤:
S1. 抽取供体小鼠的外周血20ml,采用淋巴细胞分离液分离外周血单个核细胞;
S2. 染色标记CD3+CD4-CD8-T细胞(简称为DN T细胞);
S3. 流式分选仪分选纯化DN T细胞;
S4. 加入培养基和白介素-2(20ng/ml)体外培养14天,每隔3天换一次培养基和白介素-2;
S5. 生理盐水洗涤细胞3次,静脉注射至受体(IBD小鼠模型)体内。
S6. 疗效判定:以不注射细胞、向受体内注射PBS为对照,检测受体肠道病理情况和体重下降情况。
小鼠肠道病理切片的结果如图1,从图1中可以看出,与输入PBS组相比,回输组的肠道病理情况得到了明显改善。
小鼠体重下降情况的结果如图2,图2的左图为小鼠肠道长度统计结果,图2的右图为小鼠的体重统计结果,从图2可以看出,与输入PBS组相比,DNT组(即回输组)小鼠的体重下降缓解,且肠道长度较长。
流式细胞分选结果如图3,从图3可以看出,小鼠肠道中来自供体小鼠的DN T细胞可分泌细胞因子IL-4保护肠道。

Claims (6)

1.CD4-CD8-双阴性的T细胞在制备用于治疗炎症性肠病的药物中的应用,其特征在于,所述药物包括CD4-CD8-双阴性的T细胞。
2.根据权利要求1所述的应用,其特征在于,所述CD4-CD8-双阴性的T细胞为CD3+CD4-CD8-双阴性T细胞。
3.根据权利要求2所述的应用,其特征在于,所述CD3+CD4-CD8-双阴性T细胞来自外周血。
4.根据权利要求3所述的应用,其特征在于,所述外周血为自体外周血。
5.根据权利要求4所述的应用,其特征在于,所述自体外周血的CD3+CD4-CD8-双阴性T细胞通过流式分选方法获得,同时经过体外扩增培养。
6.根据权利要求5所述的应用,其特征在于,所述体外扩增培养过程中加入白介素-2。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101252941A (zh) * 2005-08-31 2008-08-27 Ith免疫治疗控股股份公司 治疗炎性肠病
CN101945666A (zh) * 2007-12-19 2011-01-12 Mivac发展股份公司 治疗自身免疫和过敏性疾病的组合物及方法
CN108367012A (zh) * 2015-09-21 2018-08-03 美国普瑞德制药有限公司 自体免疫性和自身炎症性疾病的治疗

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AU2003249247A1 (en) * 2002-07-15 2004-02-02 Mayo Foundation For Medical Education And Research Treatment and prophylaxis with 4-1bb-binding agents
CA2629532C (en) * 2005-11-18 2016-08-09 University Health Network Method of expanding double negative t cells
CN103608452A (zh) * 2011-03-25 2014-02-26 特克赛尔公司 调节性t细胞在治疗中的使用方法
CN105483083B (zh) * 2016-01-20 2018-10-23 北京医明佳和生物科技有限公司 双阴性t细胞的转化扩增方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101252941A (zh) * 2005-08-31 2008-08-27 Ith免疫治疗控股股份公司 治疗炎性肠病
CN101945666A (zh) * 2007-12-19 2011-01-12 Mivac发展股份公司 治疗自身免疫和过敏性疾病的组合物及方法
CN108367012A (zh) * 2015-09-21 2018-08-03 美国普瑞德制药有限公司 自体免疫性和自身炎症性疾病的治疗

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