CN108220237B - 一种人的nk/t细胞系 - Google Patents
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Abstract
本发明涉及细胞系，具体公开了一种人的NK/T细胞系，保藏编号为CGMCC No.14892。本发明所提供的人NK/T细胞系可长期传代，大量扩增，并具有较强的抗肿瘤活性，为研究者深入开展慢性活动性EBV感染疾病机理研究、筛选慢性活动性EBV感染、EBV相关淋巴细胞增殖症等诊断分子标记物、治疗预后标记物及治疗药物、CD8+T细胞、自然杀伤细胞抗肿瘤作用机制及临床过继免疫治疗提供新的实验模型。
Description
技术领域
本发明涉及细胞系，具体地说，涉及一种人的NK/T细胞系。
背景技术
T细胞可以根据是否表达CD4或CD8受体分子而划分为两个亚群。CD8+T细胞通过直接杀伤作用，主要负责对靶细胞的清除，如被病毒感染的细胞和表达有肿瘤特异性抗原的细胞。自然杀伤细胞(NK细胞)是机体重要的免疫细胞，具有抗肿瘤、抗病毒感染和免疫调节作用。
NK细胞和T细胞一直被认为是杀死癌细胞和治疗感染的绝佳武器。过继免疫治疗通常需要从癌症病人体内收集T细胞，在实验室给它们武装上肿瘤特异性受体，并将之回输进入病人体内。然而，过继免疫治疗很耗时，同时癌症病人可能没有足够的T细胞或者NK细胞可用。同时NK细胞和T细胞纯化和体外大量扩增技术上存在一定难度。因此科学家试图建立细胞系，用同种异体NK细胞进行肿瘤生物治疗，国外科学家相继建立了HANK、KHYG-1、NK-92、NKL、PLT、MOTN、IMC-1、NK-YS、KAI3、SNK-1、SNK6以及YT、NK3.3等，还有通过稳定转染IL-2获得的NK-92MI、IL-2/NKL、IL-2/NK3.3等转基因NK/T细胞系。尤其是NK-92的建立，促进了其在肿瘤过继免疫治疗中的应用，并为深入开展研究NK和或T细胞的特征、功能、进一步揭示它在免疫系统中的确切作用，提供了有力的研究材料。
现有技术中，公开了众多由来自鼻腔T/NK细胞淋巴瘤患者(Nagata H,Konno A,Kimura N,Zhang Y,Kimura M,Demachi A,Sekine T,Yamamoto K,Shimizu N.Blood 2001,97:708–713)以及CAEBV患者的外周血单个核细胞所建立的NK/T细胞系或T细胞系(HiroshiNagata,1Tsutomu Numata,1Akiyoshi Konno,Pathology International 2001；51:778–785)。这些细胞系是用超过10mL的外周血，经过流式细胞分选后利用高浓度的细胞因子培养获得的纯化的细胞系。这类细胞系依赖高浓度的细胞因子，事先经过纯化，研究表明这类细胞不能完全代表CAEBV的发病模型(Ken-Ichi Imadome1,Misako Yajima1,Ayako Arai，et al.Plos Pathogen,2011,V7(10),e1002326)。因此，提供一种来源CAEBV患者的未经纯化的，包含患者自身感染NK和或T的细胞系，对于研究该类疾病和筛选诊断分子marker以及筛选药物具有重要意义和独特价值。
发明内容
为了解决现有技术中存在的问题，本发明的目的是提供一株可长期在体外传代培养，大量扩增的能作为研究模型的人NK/T细胞系。
为了实现本发明目的，本发明的技术方案如下：
一种人NK/T细胞系，经鉴定为人NK/T细胞系L311，分类命名为：人的细胞系，保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮政编码：100101)，保藏日期为2017年12月4日，保藏编号为CGMCC No.14892。
本发明所提供的人NK/T细胞系可长期传代，大量扩增，为研究者深入开展慢性活动性EBV感染疾病机理研究、筛选慢性活动性EBV感染、EBV相关淋巴细胞增殖症等治疗药物、CD8+T细胞、自然杀伤细胞抗肿瘤作用机制及临床过继免疫治疗提供新的实验模型。
本发明所述的人NK/T细胞系L311，是一株源自慢性活动性EBV感染淋巴细胞增殖症病人的细胞系。
本发明进一步提供了所述细胞系在进行慢性活动性EBV感染、EBV相关淋巴细胞增殖症病人、淋巴瘤或白血病病人疾病致病机理研究方面的应用。
本发明还提供了所述细胞系在筛选诊断EBV相关淋巴细胞增殖症、淋巴瘤或白血病分子标记中的应用，以及在筛选治疗EBV相关淋巴细胞增殖症、淋巴瘤或白血病病人分子靶标中的应用。
本发明还提供了所述细胞系在筛选治疗EBV相关淋巴细胞增殖症、淋巴瘤或白血病病人敏感药物中的用途。
进一步地，本发明所述细胞系具有较强的抗肿瘤活性，本发明还提供了一种利用所述L311细胞系预防/治疗癌症、淋巴瘤、淋巴细胞增殖症的方法。
所述方法包括：
(1)将有效量的L311细胞向受试者进行给药。
(2)利用生物技术、基因编辑等手段改造L311细胞，将之向受试者进行给药。
(3)将L311细胞进行纯化、改造后向受试者给药。
本发明还提供了从所述人NK/T细胞系L311中分选纯化的细胞组分，以及含有所述细胞组分的混合物。
本发明涉及到的原料或试剂均为普通市售产品，涉及到的操作如无特殊说明均为本领域常规操作。
在符合本领域常识的基础上，上述各优选条件，可以相互组合，得到具体实施方式。
本发明的有益效果在于：
本发明提供了一种来源于慢性活动性EBV患者的NK/T细胞系，可长期传代，大量扩增，并具有较强的抗肿瘤活性，为研究者深入开展慢性活动性EBV感染疾病机理研究、筛选慢性活动性EBV感染、EBV相关淋巴细胞增殖症等治疗药物、CD8+T细胞和自然杀伤细胞抗肿瘤作用机制及临床过继免疫治疗提供新的实验模型。
附图说明
图1为本发明所述的L311细胞系在显微镜下的活细胞形态。
图2本发明所述的L311细胞CD分化抗原表型分析。
图3为本发明所述的L311细胞对鼻咽癌细胞的细胞生长抑制率，效靶比为20:1。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
1、L311细胞系的建立
1.1 标本的来源：
取自一名患有慢性活动性EBV感染女性患者的外周血2ml，肝素抗凝。并经患者及家属同意，签订知情同意书。
1.2 外周血单个核细胞的分离：
无菌抽取患者外周血2ml，用淋巴细胞分离液进行密度梯度离心，2000转/分，20分钟离心，室温20℃。吸取中界层面的单个核细胞，经PBS液洗2遍，1500转/分，10分钟，室温20度。加新鲜完全的1640培养液，将细胞悬液2m1分装置24孔培养板孔内，在37℃、5％CO2培养箱中培养，并添加50～700U的重组人IL-2。
1.3 细胞的培养建系
细胞培养72小时后，镜下观察可见透亮而生长旺盛的淋巴细胞，间隔2～3天吸掉500μl上清，加入含有重组人IL-2的1640培养基。一周以后，在镜下观察，可见细胞培养板孔内细胞生长特别旺盛，有细胞成团生长现象，周边有许多透亮圆形、大小不等的淋巴细胞密集生长，有的凝聚成葡萄串样生长。培养2周左右，仔细挑选出几个细胞生长特别旺盛的孔，将孔内细胞轻轻吹打，将细胞传代至T25细胞瓶中培养，随后细胞生长状况好，且生长速率快，2-3天可以传代一次。间隔2-3天显微镜观察细胞形态：取细胞培养板置于倒置显微镜下，目镜10×，物镜20×。图1所示为L311细胞形态(200×)，细胞呈透亮圆形，生长旺盛。
该细胞已连续稳定生长3个月以上，经液氮冻存后复苏，细胞状态好，存活率达95％以上，将该细胞命名为L311细胞系，保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮政编码：100101)，保藏日期为2017年12月4日，保藏编号为CGMCC No.14892。
L311细胞培养所用的培养液为：1640培养液(life产品)，内含20％人血清(MRC产品，56℃灭能处理30分钟)、rIL-2(Roche)50-700U/ml并加适量双抗(life产品)。
2、细胞表型分析
细胞表面CD分化抗原分析
取约1×106对数生长期的传代细胞，收集于15ml的离心管中，用台盼兰活细胞计数，细胞经PBSA液(PBS加2％FBS)洗2遍后，加入200μl人血清孵育10min，2000rpm离心2min，收集于1.5ml的离心管用100μl PBS，分成5管，2000rpm离心2min去除上清，加入20μl相应的CD抗体，避光冰上放置30分钟。细胞经PBSA液(PBS加2％FBS)洗2遍后，再加400μl PBSA液，经流式细胞仪(FACS美国BD公司)上样检测。
图2所示L311表型分析结果CD19-HLA-DR+CD16+CD56+CD3+CD4-CD8+。其中主要为CD8+T细胞。
3、细胞体外对3株肿瘤细胞的杀伤作用研究
L311细胞及3株鼻咽癌细胞系，经传代培养，细胞生长稳定后，取对数生长期的细胞，经离心收集细胞，再用PBS液洗一次，计数，新建细胞定量至1×105/ml的细胞悬液备用，3株肿瘤细胞按效靶比20∶1进行稀释。按组别取效应细胞100进行，约2×104细胞/孔，靶细胞100μl细胞铺于96孔板中，总量200μl，效应细胞及靶细胞均设对照组。每组设3个复孔。置于37℃CO2培养箱中培养，分别取实验后1、3、5、7、9天的细胞，加入CCK-8，10μl/孔，加完后轻轻敲击培养板以帮助混匀。37℃孵育1-3小时后，在酶联免疫测定仪上检测吸光度(OD值)，采用双波长测定，检测波长450nm，参比波长630nm。经重复实验，取平均值。检测新建细胞对3株肿瘤细胞的杀伤作用情况，实验结果如图3所示。
根据OD值按下列公式计算细胞生长抑制率，由图3可以看出，效靶比20∶1均可见明显抗瘤活性，尤其以对鼻咽癌细胞系CNE2和Hone1的细胞生长抑制作用显著。
细胞生长抑制率(IR)＝(阴性对照组平均OD值-处理组平均OD值)/(阴性对照组平均OD值-空白组OD值)×100％。
其中，空白组为：培养基+CCK-8无细胞孔。
应当理解的是，对上述实施例所用试剂或原料的用量进行等比例扩大或者缩小后的技术方案，与上述实施例的实质相同。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (3)
1.一种人NK/T细胞系L311，其保藏号为CGMCC NO.14892。
2.权利要求1所述的人NK/T细胞系L311在制备用于治疗鼻咽癌的药物中的应用。
3.含有权利要求1所述的人NK/T细胞系L311的组合物。
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