CN116254234A - 基因修饰的k562细胞及其在体外培养nk细胞中的应用 - Google Patents
基因修饰的k562细胞及其在体外培养nk细胞中的应用 Download PDFInfo
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Abstract
本发明公开了一种基因修饰的K562细胞，其基因组中包含有CD48、CD137L、CD64、tCD19、IL‑21的编码基因，其构建方法为：(1)取CD48、CD137L、tCD19、CD64的基因片段，连接插入慢病毒表达载体，感染K562细胞，得到稳定表达CD48、CD137L、tCD19、CD64的K562细胞；(2)取IL‑21的基因片段，连接插入慢病毒表达载体，感染上述稳定表达的K562细胞，得到稳定表达CD48、CD137L、tCD19、CD64、IL‑21的K562细胞。所述基因修饰的K562细胞作为滋养层细胞在体外培养NK细胞中的应用。本发明构建的稳转细胞株可以使NK细胞快速增殖，且NK细胞纯度高，杀伤力强。
Description
技术领域
本发明涉及一种基因修饰的K562细胞及其在体外培养NK细胞中的应用，属于NK细胞培养技术领域。
背景技术
过继性免疫治疗(adoptive cell therapy，ACT)是将患者的淋巴细胞从体内分离出来，在体外经过激活、扩增培养、筛选或者修饰后，将具有杀伤肿瘤能力的淋巴细胞再回输到患者体内，用来控制和清除肿瘤的一种治疗方式，也是目前肿瘤免疫治疗领域最具有潜力的一种治疗方式。自然杀伤细胞(NK细胞)是不同于T细胞、B细胞的第三类淋巴细胞，属固有免疫系统，广泛存在于外周血、骨髓及组织中，约占外周血淋巴细胞总数的10％～20％，脐带血淋巴细胞的5％。NK细胞具有独特的肿瘤识别机制，可以通过释放含有穿孔素和颗粒酶的细胞质颗粒直接杀伤靶细胞，释放细胞因子诱导肿瘤细胞凋亡，介导ADCC作用杀伤靶细胞。NK细胞不表达抗原特异性识别受体，不会导致免疫排斥反应，是理想的同种异体治疗载体。通过嵌合抗原受体(CAR)修饰的NK细胞发挥更强的抗肿瘤活力，且不会出现GvHD、CRS，具有毒副作用小等优点，可作为现货细胞治疗产品，在细胞治疗领域具有显著优势。
目前，NK细胞的体外扩增方式主要有两种。一种是通过IL-2、IL-12、IL-15、IL-18、IL-21等细胞因子对NK细胞刺激扩增，但这些细胞因子诱导的NK细胞体外增殖能力一般是有限的，成本较高，纯度却较低。另一种是通过滋养细胞刺激NK细胞增殖，如Wilms肿瘤细胞系、自体PBMCs、EBV转化的淋巴母细胞、慢性粒细胞白血病细胞系K562细胞，但在增殖速度方面尚有改进的空间，且所得NK细胞的杀伤力不强。
发明内容
针对上述现有技术，本发明提供了一种基因修饰的K562细胞——同时表达CD48、CD137L、CD64、tCD19、IL-21的K562细胞，并将其作为NK细胞的滋养层细胞进行应用。
本发明是通过以下技术方案实现的：
一种基因修饰的K562细胞，其基因组中包含有CD48、CD137L、CD64、tCD19、IL-21的编码基因。
所述基因修饰的K562细胞的构建方法，包括以下步骤：
(1)取CD48、CD137L、tCD19、CD64的基因片段，连接插入慢病毒表达载体，感染K562细胞，得到稳定表达CD48、CD137L、tCD19、CD64的K562细胞；
(2)取IL-21的基因片段，连接插入慢病毒表达载体，感染上述稳定表达CD48、CD137L、tCD19、CD64的K562细胞，得到稳定表达CD48、CD137L、tCD19、CD64、IL-21的K562细胞；
(3)将步骤(2)所得K562细胞扩大培养后，用100Gy的γ射线辐照培养液，该培养液即可作为滋养层细胞，用于NK细胞的体外培养。
所述基因修饰的K562细胞作为滋养层细胞在体外培养NK细胞中的应用。
一种体外培养NK细胞的方法：将包含有上述基因修饰的K562细胞的培养液，与NK细胞共同置于培养基中，补充200U/mL的IL-2，培养7～21天，间隔一天补充新鲜培养基，并补充200U/mL的IL-2。
进一步地，基因修饰的K562细胞与NK细胞的数量比例为2:1。
本发明利用CD48、CD137L、CD64、CD19、IL-21完整或具备其功能的基因片段，构建表达载体，包装慢病毒，然后通过慢病毒感染K562细胞的方式，使各靶蛋白可以在K562细胞中稳定表达。再利用γ射线辐照使其失去增殖功能，但是保留活性。将筛选得到的稳转细胞株K562-CD48-CD137L-tCD19-CD64-mbIL21(1D8-IL21)与NK细胞混合，进行体外扩增，并同时补充细胞因子IL-2，过程中应用AOPI技术方法检测NK细胞增殖、流式细胞术检测NK纯度、利用荧光素酶检测杀伤性、ELISA试剂盒检测细胞因子分泌，测试本发明的滋养层细胞对NK细胞的增殖、杀伤、纯度、细胞因子分泌等方面的影响。结果显示：本发明构建的稳转细胞株K562-CD48-CD137L-tCD19-CD64-mbIL21(1D8-IL21)，可以使NK细胞快速增殖，且NK细胞纯度高，杀伤力强。
附图说明
图1：K562-CD48-CD137L-tCD19-CD64-mbIL21滋养细胞模式图。
图2：滋养细胞流式检测结果示意图。
图3：NK细胞增殖曲线示意图。
图4：NK细胞纯度检测结果示意图。
图5：杀伤性能结果示意图。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而，本发明的范围并不限于下述实施例。本领域技术人员能够理解，在不背离本发明的精神和范围的前提下，可以对本发明进行各种变化和修饰。
下述实施例中所涉及的仪器、试剂、材料，若无特别说明，均为现有技术中已有的常规仪器、试剂、材料，可通过正规商业途径获得。下述实施例中所涉及的实验方法、检测方法，若无特别说明，均为现有技术中已有的常规实验方法、检测方法。
实施例1滋养细胞的制备
1.1滋养细胞细胞株的构建(模式如图1所示)
NCBI中查询CD48、CD137L、CD19、CD64功能基因编码序列，其中CD19需要胞内段截短(不含胞内信号传导区域)，通过基因合成方法合成CD48、CD137L、tCD19、CD64基因片段，然后在首尾引入不同的酶切位点，连接插入慢病毒表达载体，进而利用Lipofectamine2000转染试剂与包装质粒PMD2G\psPAX2包装慢病毒，感染K562细胞。流式细胞检测感染K562后各靶标的表达，通过有限稀释方法挑选可同时表达各靶标的单克隆细胞，使得CD48、CD137L、tCD19、CD64稳定表达在K562细胞上。
在K562-CD48-CD137L-tCD19-CD64基础上导入可在细胞膜表面稳定表达IL-21的mbIL21基因片段，其所表达的IL-21(mbIL21)由GM-CSF信号肽、IL-21、lgG4铰链区、lgG4Fc、CD4跨膜区组成，mbIL21通过慢病毒感染的方法使其稳定表达在K562-CD48-CD137L-tCD19-CD64细胞株上，并利用流式细胞鉴定，确保所有靶标均表达。最终得到了稳定表达的K562-CD48-CD137L-tCD19-CD64-mbIL21细胞株，所有靶标的表达阳性率在90％以上，检测结果如图2所示，提示滋养细胞株构建成功。
1.2滋养细胞株应用处理
将上述K562-CD48-CD137L-tCD19-CD64-mbIL21扩大培养后，用100Gy的γ射线进行辐照，辐照后细胞冻存于液氮备用。
实施例2NK细胞的筛选
2.1利用淋巴细胞分离液进行分离脐血来源的单个核细胞，然后进行细胞计数，300g离心，10min。
2.2用缓冲液重悬细胞，40μL/107个细胞；每107个细胞加入10μL的NK CellBiotin-Antibody Cocktail；混匀后，置于2～8℃，5分钟；每107个细胞补加30μL缓冲液。
2.3每107个细胞加入20μL的NK cell microbead cocktail；混匀后，置于2～8℃，10分钟。
2.4将分选柱放置于分选磁铁上，利用适量缓冲液润洗柱子。
2.5缓冲液流完后，将以上细胞悬液加入到柱子中，收集流出液(分选后的NK细胞)；以适当体积的缓冲液清洗一次柱子，并收集流出液。
实施例3NK细胞的扩增
将上述分选得到的NK细胞重悬于NK-MACS培养基(额外添加10％的FBS)中。取辐照后的滋养细胞(实施例1制备)，重悬在该NK培养基中，与NK细胞比例按照2:1混匀，并补充200U/mL的IL-2，继续培养，间隔一天补充新鲜培养基(含200U/mL的IL-2)。培养期间利用AOPI进行细胞增殖计数，监控细胞增殖曲线。培养结束，用CD56/CD3抗体标记，流式细胞术检测收集的细胞纯度。
增殖曲线如图3所示，细胞纯度检测结果如图4所示。实施例1所构建的滋养细胞(1D8-mbIL21)可使NK细胞增殖倍数达到1000倍以上，且优于商品化的滋养细胞(PC)，NK细胞中CD56+CD3-细胞比例在约99％。
实施例4NK细胞杀伤实验
以稳转Luciferase的Raji细胞作为靶细胞，将生长状态良好的NK细胞重悬在培养基中，与靶细胞Raji-Luciferase以1:1、5:1、10:1接种到96孔板中，共培养4h，Bright-GloTM荧光素酶测定试剂盒，检测靶细胞Luciferase释放水平，检测NK细胞杀伤能力。结果如图5所示，与商品化的滋养细胞(PC)所扩增的NK细胞相比，本发明构建的滋养细胞K562-CD48-CD137L-tCD19-CD64-mbIL21(1D8-mbIL21)所扩增的NK细胞具有相同的杀伤靶细胞能力。
给本领域技术人员提供上述实施例，以完全公开和描述如何实施和使用所主张的实施方案，而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权利要求的范围内。
Claims (5)
1.一种基因修饰的K562细胞，其特征在于：其基因组中包含有CD48、CD137L、CD64、tCD19、IL-21的编码基因。
2.权利要求1所述的基因修饰的K562细胞的构建方法，其特征在于，包括以下步骤：
(1)取CD48、CD137L、tCD19、CD64的基因片段，连接插入慢病毒表达载体，感染K562细胞，得到稳定表达CD48、CD137L、tCD19、CD64的K562细胞；
(2)取IL-21的基因片段，连接插入慢病毒表达载体，感染上述稳定表达CD48、CD137L、tCD19、CD64的K562细胞，得到稳定表达CD48、CD137L、tCD19、CD64、IL-21的K562细胞。
3.权利要求1所述的基因修饰的K562细胞作为滋养层细胞在体外培养NK细胞中的应用。
4.一种体外培养NK细胞的方法，其特征在于：将包含有权利要求1所述的基因修饰的K562细胞的培养液，与NK细胞共同置于培养基中，补充200U/mL的IL-2，培养7～21天，间隔一天补充新鲜培养基，并补充200U/mL的IL-2。
5.根据权利要求4所述的体外培养NK细胞的方法，其特征在于：所述基因修饰的K562细胞与NK细胞的数量比例为2:1。
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