CN111973585B - 银杏双黄酮在制备抗髓系抑制性细胞的药物中的用途 - Google Patents
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Abstract
本发明公开了银杏双黄酮在制备抗髓系抑制性细胞的药物中的用途。本申请发明人经过研究发现，银杏双黄酮能明显抑制髓系抑制性细胞的产生，可用于制备抗髓系抑制性细胞的药物。本发明提供了银杏双黄酮的药物新用途，同时为制备抗髓系抑制性细胞的药物提供了新的选择。
Description
技术领域
本发明属于生物医药技术领域，具体涉及银杏双黄酮在制备抗髓系抑制性细胞的药物中的用途。
背景技术
髓系抑制性细胞(Myeloid-derived suppressor cells，MDSCs)是骨髓来源的一群异质性细胞，是树突状细胞(dendritic cells，DCs)、巨噬细胞和(或)粒细胞的前体，具有显著抑制免疫细胞应答的能力。MDSCs来源于骨髓祖细胞和未成熟髓细胞(immaturemyeloid cells，IMCs)。正常情况下，是树突状细胞(DC)、巨噬细胞和粒细胞的前体，能迅速地分化为成熟的粒细胞、DCs和巨噬细胞，并进入相应的器官、组织，发挥正常免疫功能，IMCs占外周血单个核细胞的0.5％左右。在肿瘤、感染、炎症、败血症、外科损伤等其它病理条件下，受细胞因子的作用，这些髓系来源的前体细胞成熟受阻，因而停留在各个分化阶段，成为具有免疫抑制功能的MDSCs。它们并在细胞因子的作用下被募集、迁移、扩增，使得其在外周血中的数量和比例增加十倍左右，约占患者外周血单个核细胞(PBMC)的10％，贯穿疾病发生的整个过程。MDSCs主要分为两个亚群：粒细胞PMN-MDSCs和单核M-MDSCs。在大多数类型的癌症中，PMN-MDSCs占全部MDSCs的70-80％，而M-MDSCs通常不超过20％。
银杏双黄酮，银杏科植物银杏的干燥叶提取物，现有研究表明可增加脑血管流量，降低脑血管阻力，改善脑血管循环功能，保护脑细胞，免受缺血损害，扩张冠状动脉，防止心绞痛及心肌梗塞，抑制血小板聚集，防止血栓形成，清除有害的氧化自由基，提高免疫能力，具有防癌抗衰功能。对治疗冠心病、心绞痛、脑动脉硬化、老年性痴呆、高血压等病有很好的疗效。
目前的研究表明MDSCs与肿瘤、自身免疫性疾病、慢性感染、创伤和炎症等多种疾病的发生发展密切相关。
发明内容
基于此，本发明提供了银杏双黄酮的药物新用途，银杏双黄酮能有效抑制髓系抑制性细胞的产生，可用于制备抗髓系抑制性细胞的药物。
为实现上述目的，本发明采用的技术方案为：
银杏双黄酮在制备抗髓系抑制性细胞的药物中的用途。
优选地，所述抗髓系抑制性细胞的药物可防治肿瘤、自身免疫性疾病、慢性感染或炎症。
优选地，所述肿瘤为胃癌。
本发明还提供了一种抗髓系抑制性细胞的药物。
为实现上述目的，本发明采用的技术方案为：
一种抗髓系抑制性细胞的药物，所述药物的活性成分包含银杏双黄酮。
银杏双黄酮作为活性成分，可与药物学可接受的载体一起，制备得到抗髓系抑制性细胞的药物。
优选地，所述药物可防治肿瘤、自身免疫性疾病、慢性感染或炎症。
优选地，所述肿瘤为胃癌。
优选地，所述药物的剂型为片剂、颗粒剂、溶液剂、粉剂、胶囊剂、注射液或粉针剂。
相对于现有技术，本发明的有益效果为：发明人经过大量的研究发现，银杏双黄酮能显著抑制小鼠以及人MDSCs的产生，可用于制备抗MDSCs的药物。MDSCs是一种具有显著抑制免疫细胞应答能力的细胞，与多种疾病的发生和发展密切相关，包括肿瘤、自身免疫性疾病、慢性感染、创伤和炎症等疾病。因此，抑制MDSCs的药物可用于防治肿瘤、自身免疫性疾病、慢性感染、创伤和炎症等疾病。
附图说明
图1为银杏双黄酮抑制小鼠MDSCs的流式检测结果图。
图2为银杏双黄酮抑制人MDSCs的流式检测结果图。
具体实施方式
为了便于理解本发明，下面将参照实施例对本发明进行更全面的描述，以下给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。应理解，下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用试剂，均为市售产品。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
实施例1银杏双黄酮抑制小鼠MDSCs
1、实验方法
(1)从小鼠骨髓中分离骨髓细胞：
1)颈椎脱位法处死8周龄C57小鼠，将小鼠浸泡在75％酒精中5min；取出小鼠，用眼科剪剪开小鼠双侧腿部皮肤，自髋关节剪下双侧腿骨；小心剪去股骨与胫骨的附着肌肉，勿伤及关节面，忌暴露骨髓腔；
2)将剥离出的股骨、胫骨浸泡于75％酒精中3min后转移至预冷的RPMI1640培养基中；
3)用眼科剪轻轻剪开股骨或胫骨两端，然后用5ml无菌注射器吸取RPMI1640培养基，对准50ml离心管冲出骨髓腔中骨髓组织；
4)3000rpm/min，室温离心5min，弃上清，加入5ml红细胞裂解液ACK，吹打混匀后裂解5min，然后加入30ml预冷的PBS终止裂解；室温3000rpm/min，离心5min以获取细胞沉淀；
5)弃上清后再加入10ml预冷的RPMI 1640，吹打重悬细胞，显微镜细胞计数板计数，备用。
(2)对步骤(1)获得的骨髓细胞进行体外培养：
1)细胞计数后，用RPMI1640+10％FBS+100U/ml青霉素+0.1mg/ml链霉素+50uMβ-ME的完全培养基将细胞密度调整至1×106cells/ml；
2)培养体系中加入重组小鼠源GM-CSF细胞因子(20ng/ml)，充分混匀后，将细胞悬液按每孔1ml铺于48孔板中。
(3)银杏双黄酮处理骨髓细胞
向步骤(2)所述培养体系中加入银杏双黄酮(1.5umol/ml)处理，继续进行体外培养6天，DMSO处理组作为对照。
(4)流式细胞术检测：
体外培养6天后，收集细胞进行流式细胞术检测：
1)取含0.5million的单细胞悬液，加入5ml PBS混匀，3000rpm/min离心5min，弃尽上清；
2)用于分析小鼠MDSC的流式抗体染色组合为：PMN-MDSC(CD11b+Gr1+Ly6C-Ly6Ghi)和M-MDSC(CD11b+Gr1+Ly6ChigLy6G-)；
3)按照抗体说明书，抗体1:200使用PBS稀释，每个样本用100μl抗体稀释液。将流式管于振荡器上振荡，充分混匀；于4℃冰箱避光染色30min。加入4ml PBS，3000rpm/min离心5min，弃去上清；加入500μl PBS重悬细胞后，流式上机检测，获取数据并用FlowJo10软件进行分析。
2、实验结果
结果如图1所示，银杏双黄酮能显著抑制小鼠MDSCs的产生，使培养体系中MDSCs的比例下降，PMN-MDSCs和M-MDSC两种亚型均受到抑制。
实施例2银杏双黄酮抑制人MDSCs
1、实验方法
(1)从人外周血中分离单核细胞：
取健康志愿者外周血5ml，分离得到单个核细胞：
1)用含EDTA抗凝采血管收集健康志愿者外周血，然后加入等3倍体积的PBS溶液，轻轻混匀；
2)向50ml无菌离心管中加入3ml淋巴细胞分离液；然后用吸管将血稀释液沿着管壁缓慢的加至淋巴细胞分离液的上层，于18℃下600g(升5，降0)，离心22min；
3)离心后，用移液枪将中间白膜层吸出，放于一干净的15ml离心管中，加入10ml预冷的PBS溶液，混匀；室温下，600g离心10min，弃尽上清；
4)加入1ml ACK红细胞裂解液进行裂红，3min后，加入10ml预冷的PBS进行终止；室温下，600g离心10min，弃尽上清；
5)用5ml PBS进行重悬细胞沉淀，进行细胞计数，放冰上备用。
(2)对步骤(1)获得的单核细胞进行体外培养：
1)细胞计数后，用RPMI1640+10％FBS+100U/ml青霉素+0.1mg/ml链霉素+50uMβ-ME的完全完全培养基将细胞密度调整至1×106cells/ml；
2)培养体系中加入重组人源GM-CSF细胞因子(20ng/ml)，充分混匀后，将细胞悬液按每孔1ml铺于48孔板中；
(3)银杏双黄酮处理单核细胞
向步骤(2)所述培养体系中加入银杏双黄酮(1.5umol/ml)处理，续进行体外培养6天，DMSO处理组作为对照。
(4)流式细胞术检测：
体外培养6天后，收集细胞进行流式细胞术检测：
1)取含0.5million的单细胞悬液，加入5ml PBS混匀，3000rpm/min离心5min，弃尽上清；
2)用于分析人MDSC的流式抗体染色组合为：M-MDSC(HLA-DR-/low CD11bhiCD33hi)和PMN-MDSC(HLA-DR-/lowCD11bmedCD33med)。
3)按照抗体说明书，抗体1:200使用PBS稀释，每个样本用100μl抗体稀释液。将流式管于振荡器上振荡，充分混匀；于4℃冰箱避光染色30min。加入4ml PBS，3000rpm/min离心5min，弃去上清；加入500μl PBS重悬细胞后，流式上机检测，获取数据并用FlowJo10软件进行分析。
2、实验结果
结果如图2所示，银杏双黄酮能显著抑制人MDSCs的产生，使培养体系中MDSCs的比例下降，尤其是使PMN-MDSCs受到抑制。
上述结果表明，银杏双黄酮能显著抑制MDSCs的产生，可用于制备抗MDSCs的药物。
最后所应当说明的是，以上实施例仅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等同替换，而不脱离本发明技术方案的实质和范围。
Claims (2)
1.银杏双黄酮在制备抑制髓系抑制性细胞体外扩增的试剂中的应用。
2.根据权利要求1所述的应用，其特征在于，所述银杏双黄酮在髓系抑制性细胞体外培养体系中的浓度为1.5umol/ml。
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| CN105193784A (zh) * | 2015-10-20 | 2015-12-30 | 吉林大学 | 银杏素在制备治疗猪链球菌感染药物中的应用 |
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| CN105193784A (zh) * | 2015-10-20 | 2015-12-30 | 吉林大学 | 银杏素在制备治疗猪链球菌感染药物中的应用 |
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