CN116970561B - 一种工程化的γδT细胞及其制备方法 - Google Patents
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Abstract
本发明提出了一种工程化的γδT细胞及其制备方法，属于γδT细胞技术领域。制备外周血人单个核细胞后，经过初步扩增和纯化，获得γδT细胞，加入含有白介素组合物、唑来膦酸、经60Gy60Co照射的Daudi和XG‑7细胞的500U/ml的rhIL‑2的RPMI 1640营养液中，低频超声波培养，调整细胞密度在106‑107个细胞/mL，加入扩增因子，继续培养，获得工程化的γδT细胞。本发明工程化的γδT细胞的制备成本低廉、工序简单、条件易控、易于规模化生产，制得的工程化的γδT细胞具有很好的抗肿瘤作用，且能够工程化保存和应用，具有广阔的应用前景。
Description
技术领域
本发明涉及γδT细胞技术领域，具体涉及一种工程化的γδT细胞及其制备方法。
背景技术
治疗恶性肿瘤的传统手段有手术治疗、放射治疗和化学治疗。然而，手术治疗不能清除肿瘤的微转移灶，难以取得根治性疗效；放疗具有剂最依赖性毒性，无法有效清除潜在的转移灶，且部分患者因产生放射抵抗而对放疗不敏感；化疗易引起全身免疫抑制，且部分患者因化疗药物无法有效进入肿瘤组织或对化疗药物产生耐药性而影响疗效。随着对免疫学和肿瘤学研究的不断深入，人们提出了生物治疗的概念，其中过继细胞免疫治疗成为近年来免疫学研究的热点。γδT细胞能够以主要组织相容性复合物(majorhistocompatibility complex，MHC)非限制性方式识别肿瘤抗原，通过穿孔素和颗粒酶等途径杀伤肿瘤细胞，还与其他固有免疫细胞和适应性免疫细胞相互作用，辅助其发挥抗肿瘤作用。因此，γδT细胞在肿瘤免疫细胞治疗中是一种具有优势的候选细胞。
T淋巴细胞的抗原识别可通过高度多样性的异二聚体受体，T细胞受体(TCR)来实现。血液和淋巴器官中大约95％的人T细胞表达异二聚体αβTCR受体(αβT细胞谱系)。血液和淋巴器官中大约5％的人T细胞表达异二聚体γδTCR受体(γδT细胞谱系)。这些T细胞亚群可分别称为“αβ”和“γδ”T细胞。αβ和γδT细胞的功能不同。αβT细胞驱动MHC限制性过继免疫并且γδT细胞桥接先天和非MHC限制性过继免疫。当抗原呈递细胞(APC)在I/II类MHC的背景下呈递抗原时，随后发生αβT细胞的活化。树突状细胞是天然T细胞的已知最强的活化剂。两种信号对于完全αβT细胞活化是必要的：a)由MHC-肽与TCR-CD3复合物的相互作用产生的信号；和b)由T细胞上的CD28和APC上的B7家族的成员的相互作用产生的信号(共刺激信号)。与αβT细胞相比，γδT细胞能够直接识别抗原，并且不需要MHC-肽与TCR-CD3复合物的相互作用。因此，据称γδT细胞不是MHC限制性的。在内源性T细胞中，缺乏共刺激信号导致克隆无反应性。
内源性γδT细胞直接识别抗原的能力使γδT细胞成为有吸引力的治疗工具。然而，操纵和引导内源性γδT细胞的困难限制临床上患者源性的γδT细胞的治疗有用性。
发明内容
本发明的目的在于提出一种工程化的γδT细胞及其制备方法，制备成本低廉、工序简单、条件易控、易于规模化生产，制得的工程化的γδT细胞具有很好的抗肿瘤作用，且能够工程化保存和应用，具有广阔的应用前景。
本发明的技术方案是这样实现的：
本发明提供一种工程化的γδT细胞的制备方法，制备外周血人单个核细胞后，经过初步扩增和纯化，获得γδT细胞，加入含有白介素组合物、唑来膦酸、经60Gy60Co照射的Daudi和XG-7细胞的500U/ml的rhIL-2的RPMI 1640营养液中，低频超声波培养，调整细胞密度在106-107个细胞/mL，加入扩增因子，继续培养，获得工程化的γδT细胞。
作为本发明的进一步改进，包括以下步骤：
S1.外周血人单个核细胞的制备：取外周血，肝素抗凝，采用淋巴细胞分离液密度梯度离心法分离人单个核细胞，用RPMI 1640营养液洗涤，离心，获得外周血人单个核细胞；
S2.γδT细胞的初步扩增和纯化：将外周血人单个核细胞置于预先经抗CD3单抗包被的培养板内，用RPMI 1640营养液培养3-5d，收集细胞，加入抗CD4，抗CD16，抗CD56和磁珠二抗，置于磁砖上，收集获得γδT细胞；
S3.培养基的制备：将含有rhIL-2的RPMI 1640营养液中加入白介素组合物和唑来膦酸，加入经60Gy60Co照射的Daudi细胞和XG-7细胞，超声分散均匀，制得培养基；
S4.工程化的γδT细胞的制备：将步骤S3制得的γδT细胞加入步骤S3制得的培养基中，低频超声波处理，培养3-5d，调整细胞密度在106-107个细胞/mL，加入扩增因子，继续培养7-10d，获得工程化的γδT细胞。
作为本发明的进一步改进，所述rhIL-2的含量为500U/mL。
作为本发明的进一步改进，所述白介素组合物选自IL-1、IL-2、IL-6、IL-7、IL-11、IL-21中的至少两种。
作为本发明的进一步改进，所述白介素组合物为IL-2和IL-17的混合物，质量比为5-7:2。
作为本发明的进一步改进，所述白介素组合物的含量为1000-1200IU/mL；所述唑来膦酸的含量为15-20μg/mL。
作为本发明的进一步改进，所述Daudi细胞和XG-7细胞的个数比为2:0.9-1.1，所述Daudi细胞的数量在104-105个/mL。
作为本发明的进一步改进，所述低频超声波处理的条件为70-150W超声波。
作为本发明的进一步改进，所述扩增因子为植物血凝素和刀豆蛋白A的混合物，质量比为2-4:5，所述扩增因子的含量为5-10μg/mL。
本发明进一步保护一种上述的制备方法制得的工程化的γδT细胞。
本发明具有如下有益效果：本发明采用含有rhIL-2的RPMI 1640营养液中加入白介素组合物和唑来膦酸，加入经60Gy60Co照射的Daudi细胞和XG-7细胞获得的培养基，通过诱导增殖和Daudi细胞和XG-7细胞的共同刺激的方法，大大促进了γδT细胞的快速增殖，并且能维持一定的时间，具有需血量少，扩殖速度快，能在短期内获得高产量细胞的特点，这种激发增殖的γδT细胞具有很好的抗肿瘤活性，与γδT细胞参加肿瘤抗原的加工提呈有关。
本发明使用的低频超声波培养为γδT细胞提供了良好的外部环境和能量，从而能够大大促进γδT细胞的增殖；白介素组合物包括IL-2和IL-17的混合物，IL-2是免疫系统中的一类细胞生长因子，能调控免疫系统中白血球的细胞活性，促进Th0和CTL的增殖，也参与抗体反应、造血和肿瘤监视，IL-21参与调节T细胞增殖，协同IL-2促进γδT细胞的增殖、分化，两者的添加具有协同增效的作用。唑来膦酸是一种特异性地作用于骨的二磷酸化合物，刺激γδT细胞的体外扩增，增强其抗性。
本发明扩增因子为植物血凝素和刀豆蛋白A的混合物，其中植物血凝素是一种有丝分裂原，能激活小淋巴细胞转化为淋巴母细胞，继而分裂增殖，释放淋巴因子，并能提高巨噬细胞的吞噬功能。作为干扰素诱导剂可以刺激机体产生白介素-2和干扰素。还可以刺激机体产生非特异性抗体；刀豆蛋白A是一种(结合蛋白)，对T淋巴细胞有激发作用，因此，两者能够通过促进有丝分裂从而促进γδT细胞的快速稳定增殖，具有协同增效的作用。
本发明工程化的γδT细胞的制备成本低廉、工序简单、条件易控、易于规模化生产，制得的工程化的γδT细胞具有很好的抗肿瘤作用，且能够工程化保存和应用，具有广阔的应用前景。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，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
本实施例提供一种工程化的γδT细胞的制备方法，包括以下步骤：
S1.外周血人单个核细胞的制备：取外周血，肝素抗凝，采用淋巴细胞分离液密度梯度离心法分离人单个核细胞，用RPMI 1640营养液洗涤，5000r/min离心15min，获得外周血人单个核细胞；
S2.γδT细胞的初步扩增和纯化：将外周血人单个核细胞置于预先经抗CD3单抗包被的培养板内，用RPMI 1640营养液培养3d，收集细胞，加入抗CD4，抗CD16，抗CD56和磁珠二抗，置于磁砖上，收集获得γδT细胞；
S3.培养基的制备：将含有500U/mL rhIL-2的RPMI 1640营养液中加入1000IU/mL白介素组合物，15μg/mL唑来膦酸，加入经60Gy60Co照射的Daudi细胞和XG-7细胞，200W超声分散10min，制得培养基；
所述白介素组合物为IL-2和IL-17的混合物，质量比为5:2；
所述Daudi细胞和XG-7细胞的个数比为2:0.9，所述Daudi细胞的数量在104个/mL；
S4.工程化的γδT细胞的制备：将步骤S3制得的γδT细胞加入步骤S3制得的培养基中，调节细胞浓度为103个细胞/mL，70W超声波处理，培养3d，调整细胞密度在106个细胞/mL，加入5μg/mL扩增因子，继续培养7d，获得工程化的γδT细胞；
所述扩增因子为植物血凝素和刀豆蛋白A的混合物，质量比为2:5。
实施例2
本实施例提供一种工程化的γδT细胞的制备方法，包括以下步骤：
S1.外周血人单个核细胞的制备：取外周血，肝素抗凝，采用淋巴细胞分离液密度梯度离心法分离人单个核细胞，用RPMI 1640营养液洗涤，5000r/min离心15min，获得外周血人单个核细胞；
S2.γδT细胞的初步扩增和纯化：将外周血人单个核细胞置于预先经抗CD3单抗包被的培养板内，用RPMI 1640营养液培养5d，收集细胞，加入抗CD4，抗CD16，抗CD56和磁珠二抗，置于磁砖上，收集获得γδT细胞；
S3.培养基的制备：将含有500U/mL rhIL-2的RPMI 1640营养液中加入1200IU/mL白介素组合物，20μg/mL唑来膦酸，加入经60Gy60Co照射的Daudi细胞和XG-7细胞，200W超声分散10min，制得培养基；
所述白介素组合物为IL-2和IL-17的混合物，质量比为7:2；
所述Daudi细胞和XG-7细胞的个数比为2:1.1，所述Daudi细胞的数量在105个/mL；
S4.工程化的γδT细胞的制备：将步骤S3制得的γδT细胞加入步骤S3制得的培养基中，调节细胞浓度为103个细胞/mL，150W超声波处理，培养5d，调整细胞密度在107个细胞/mL，加入10μg/mL扩增因子，继续培养10d，获得工程化的γδT细胞；
所述扩增因子为植物血凝素和刀豆蛋白A的混合物，质量比为4:5。
实施例3
本实施例提供一种工程化的γδT细胞的制备方法，包括以下步骤：
S1.外周血人单个核细胞的制备：取外周血，肝素抗凝，采用淋巴细胞分离液密度梯度离心法分离人单个核细胞，用RPMI 1640营养液洗涤，5000r/min离心15min，获得外周血人单个核细胞；
S2.γδT细胞的初步扩增和纯化：将外周血人单个核细胞置于预先经抗CD3单抗包被的培养板内，用RPMI 1640营养液培养4d，收集细胞，加入抗CD4，抗CD16，抗CD56和磁珠二抗，置于磁砖上，收集获得γδT细胞；
S3.培养基的制备：将含有500U/mL rhIL-2的RPMI 1640营养液中加入1100IU/mL白介素组合物，17μg/mL唑来膦酸，加入经60Gy60Co照射的Daudi细胞和XG-7细胞，200W超声分散10min，制得培养基；
所述白介素组合物为IL-2和IL-17的混合物，质量比为6:2；
所述Daudi细胞和XG-7细胞的个数比为2:1，所述Daudi细胞的数量在105个/mL；
S4.工程化的γδT细胞的制备：将步骤S3制得的γδT细胞加入步骤S3制得的培养基中，调节细胞浓度为103个细胞/mL，120W超声波处理，培养4d，调整细胞密度在107个细胞/mL，加入7μg/mL扩增因子，继续培养9d，获得工程化的γδT细胞；
所述扩增因子为植物血凝素和刀豆蛋白A的混合物，质量比为3:5。
实施例4
与上实施例3相比，不同之处在于，白介素组合物为单一的IL-2。
实施例5
与上实施例3相比，不同之处在于，白介素组合物为单一的IL-17。
实施例6
与上实施例3相比，不同之处在于，扩增因子为单一的植物血凝素。
实施例7
与上实施例3相比，不同之处在于，扩增因子为单一的刀豆蛋白A。
对比例1
与上实施例3相比，不同之处在于，未添加白介素组合物。
对比例2
与上实施例3相比，不同之处在于，未添加扩增因子。
对比例3
与上实施例3相比，不同之处在于，未添加经60Gy60Co照射的Daudi细胞和XG-7细胞。
测试例1γδT细胞的扩增效率
计算实施例1-7和对比例1-3组中制得的工程化γδT细胞的体外扩增效率的计算：
扩增效率＝(培养后细胞总数×γδT细胞占细胞总数比例)/(外周血人单个核细胞总数×γδT细胞占外周血人单个核细胞比例)
结果见表1。
表1
| 组别 | 扩增效率(％) |
| 实施例1 | 720 |
| 实施例2 | 730 |
| 实施例3 | 750 |
| 实施例4 | 420 |
| 实施例5 | 410 |
| 实施例6 | 460 |
| 实施例7 | 480 |
| 对比例1 | 350 |
| 对比例2 | 380 |
| 对比例3 | 400 |
由上表可知，本发明实施例1-3中的方法制得的工程化的γδT细胞的扩增效率更高。
测试例2
将实施例1-7和对比例1-3制得的工程化的γδT细胞以及市售γδT细胞用作效应细胞，并密度调节为1×105个/mL以供使用；将复苏的A549细胞用作靶细胞，将密度调节至1×105个/mL，以备后用。将工程化的γδT细胞培养液100μL和A549细胞液按照有效目标比例10：1接种在96孔板上。每个96孔板分为5组，每组分为8个复孔。37℃、CO2浓度为5％，氧气浓度为20％，余量为氮气，此处％为体积百分比，在培养箱中过夜孵育24h，加入20μL/孔MTT溶液，在原条件孵育3h，离心，每个孔中添加150μL二甲基亚砜，轻轻摇动5min，晶体完全融化，然后静置5min。酶标仪测定在490nm处的吸光度(A)值。
杀伤率(％)＝〔1-(实验组OD值-单独的效应细胞组OD值)/单独的靶细胞组OD值〕×100％
结果见表2。
表2
| 组别 | 杀伤率(％) |
| 实施例1 | 91.0 |
| 实施例2 | 90.7 |
| 实施例3 | 92.6 |
| 实施例4 | 83.5 |
| 实施例5 | 84.1 |
| 实施例6 | 85.5 |
| 实施例7 | 86.0 |
| 对比例1 | 78.9 |
| 对比例2 | 82.3 |
| 对比例3 | 80.6 |
| 市售 | 73.2 |
由上表可知，本发明实施例1-3制得的工程化的γδT细胞对肺癌A549细胞具有更高的杀伤率。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (6)
1.一种工程化的γδT细胞的制备方法，其特征在于，包括以下步骤：
S1.外周血人单个核细胞的制备：取外周血，肝素抗凝，采用淋巴细胞分离液密度梯度离心法分离人单个核细胞，用RPMI 1640营养液洗涤，离心，获得外周血人单个核细胞；
S2.γδT细胞的初步扩增和纯化：将外周血人单个核细胞置于预先经抗CD3单抗包被的培养板内，用RPMI 1640营养液培养3-5d，收集细胞，加入抗CD4，抗CD16，抗CD56和磁珠二抗，置于磁砖上，收集获得γδT细胞；
S3.培养基的制备：将含有rhIL-2的RPMI 1640营养液中加入白介素组合物和唑来膦酸，加入经60Gy60Co照射的Daudi细胞和XG-7细胞，超声分散均匀，制得培养基；
所述白介素组合物为IL-2和IL-17的混合物，质量比为5-7:2；
S4.工程化的γδT细胞的制备：将步骤S3制得的γδT细胞加入步骤S3制得的培养基中，低频超声波处理，培养3-5d，调整细胞密度在106-107个细胞/mL，加入扩增因子，继续培养7-10d，获得工程化的γδT细胞；
所述扩增因子为植物血凝素和刀豆蛋白A的混合物，质量比为2-4:5，所述扩增因子的含量为5-10µg/mL。
2.根据权利要求1所述的制备方法，其特征在于，所述rhIL-2的含量为500U/mL。
3.根据权利要求1所述的制备方法，其特征在于，所述白介素组合物的含量为1000-1200IU/mL；所述唑来膦酸的含量为15-20µg/mL。
4.根据权利要求1所述的制备方法，其特征在于，所述Daudi细胞和XG-7细胞的个数比为2:0.9-1.1，所述Daudi细胞的数量在104-105个/mL。
5.根据权利要求1所述的制备方法，其特征在于，所述低频超声波处理的条件为70-150W超声波。
6.一种如权利要求1-5任一项所述的制备方法制得的工程化的γδT细胞。
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| CN113249321A (zh) * | 2021-05-31 | 2021-08-13 | 上海南滨江细胞生物科技有限公司 | 一种外周血nk细胞的培养方法 |
| CN115678846A (zh) * | 2022-09-01 | 2023-02-03 | 广东龄值生物科技有限公司 | 一种肿瘤特异性γδT细胞及其制备方法 |
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