CN113862223A - 一种能显著增强杀伤活性的nk细胞扩增方法 - Google Patents
一种能显著增强杀伤活性的nk细胞扩增方法 Download PDFInfo
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Abstract
本发明提供了一种能显著增强杀伤活性的NK细胞扩增方法,包括以下步骤:(1)构建含有NK扩增所需的细胞因子基因且带有哺乳动物启动子的重组杆状病毒;(2)利用步骤(1)制备的重组杆状病毒对MSC细胞进行感染;(3)将步骤(2)中感染后的MSC细胞加入已活化培养的NK细胞中共培养,共培养24‑60小时后,NK细胞再继续传代扩增培养。本发明扩增方法既能显著增强NK细胞杀伤活性又能大大提升扩增倍数,并且整个方法过程满足GMP要求同时也符合临床使用标准。
Description
技术领域
本发明涉及细胞生物学领域,具体涉及一种能显著增强杀伤活性的NK细胞扩增方法。
背景技术
NK细胞称自然杀伤细胞(Natural kill cell)是人体先天性免疫系统的重要组成部分,细胞表型特征CD3-CD56+,NK细胞发挥杀伤靶细作用时无需抗原预先致敏,也无MHC限制。而且NK细胞CD56分子表达的密度的差异,将NK分为CD56dim和CD56bright两个亚群,CD56dim占NK细胞的90%以上,主要为细胞毒作用,具有很强的杀伤活性;CD56bright可产生大量细胞因子,主要起免疫调节作用。
NK不同于T细胞和B细胞,缺少基因重排,不像T细胞、B细胞一样通过特异性TCR识别靶细胞,而是通过胚系基因表达的受体识别靶细胞。NK细胞受细胞表面多种受体调控:杀伤细胞活化受体(KAR)和杀伤细胞抑制受体(KIR)。正常情况下,KAR与自身细胞上多糖类抗原结合产生活化信号,同时KIR与MHCI类分子结合,产生抑制信号且占主导地位,以保证自身组织细胞不被破坏;当细胞表面MHCI类分子发生改变或减少缺失,影响与KIR结合,不能产生抑制性信号,NK则活化,产生杀伤效应。NK细胞的杀伤机理有:释放穿孔素和颗粒酶等细胞毒性颗粒,裂解肿瘤细胞;活化NK细胞表达Fas(CD95)配体和TRAIL(TNF relatedapoptosis inducing ligand)分子,诱导靶细胞进入程序死亡;NK分泌多种细胞因子介导杀伤作用;在肿瘤或病毒特异性IgG抗体存在条件下,NK细胞也可通过表面IgGFC(FCrRⅢ)介导产生ADCC效应。
人们经研究认为:NK细胞的动物实验及临床研究报告显示,NK对多种肿瘤杀伤的有效性,目前国内有200多家研究所与企业涉及NK细胞的项目研究与临床试验;在美国NK细胞临床试验研究多达800多项,目前单就NIH注册的NK细胞治疗淋巴瘤临床试验超过20余项,部分使用细胞多来源于脐血或患者家属等。
目前NK细胞规模化制备存在以下产业化瓶颈:
1、添加可溶性重组生长因子、IL-2、IL-15等常规NK细胞培养方法,存在扩增倍数有限且杀伤活性不高的缺点。
2、利用K562作为NK细胞扩增培养时的滋养层细胞(K562IL15-4-IBBligand)经伽马辐射后使用,不符合GMP标准包括安全性责疑。
因此,本领域技术人员一直致力于研究一种既能增强NK细胞杀伤活性又能大大提升扩增倍数并且能符合GMP标准和临床使用标准要求的NK细胞扩增方法。
发明内容
本发明的目的,就是为了解决上述问题而提供了一种能显著增强杀伤活性的NK细胞扩增方法,既能显著增强NK细胞杀伤活性又能大大提升扩增倍数,并且整个方法过程满足GMP要求同时也符合临床使用标准。
本发明的目的是这样实现的:
本发明提供了一种能显著增强杀伤活性的NK细胞扩增方法,包括以下步骤:
(1)构建含有NK扩增所需的细胞因子基因且带有哺乳动物启动子的重组杆状病毒;
(2)利用步骤(1)制备的重组杆状病毒对MSC细胞进行感染;
(3)将步骤(2)中感染后的MSC细胞加入已活化培养的NK细胞中共培养,共培养24-60小时后,NK细胞再继续传代扩增培养;
其中,所述步骤(1)中所述细胞因子为IL-2、IL-15、IL-21、IFN-γ中的至少一种。
上述的能显著增强杀伤活性的NK细胞扩增方法,其中,所述步骤(3)中,所述MSC细胞和NK细胞按照1:5-20的细胞数量比例混合共培养。
上述的能显著增强杀伤活性的NK细胞扩增方法,其中,所述步骤(3)中,所述MSC细胞和NK细胞按照1:8-12的细胞数量比例混合共培养。
上述的能显著增强杀伤活性的NK细胞扩增方法,其中,所述步骤(1)具体为:分别构建含IL-2、IL-15、IL-21和IFN-γ基因的质粒载体;分别用上述质粒载体制备带有哺乳动物启动子的重组杆状病毒。
上述的能显著增强杀伤活性的NK细胞扩增方法,其中,所述步骤(1)中的哺乳动物启动子为CMV。
上述的能显著增强杀伤活性的NK细胞扩增方法,其中,所述步骤(3)中所述已活化培养的NK细胞是被IL-2活化的。
作为生物技术领域的一股力量,自从1983年杆状病毒作为载体,首次在昆虫细胞中表达人类干扰素(IFN-β)(Smith等人,1983),杆状病毒-昆虫细胞表达系统已经被充分用于大量重组蛋白的生产中。其中苜蓿银纹夜蛾核多核病毒(AcMNPV)作为最经典的杆状病毒表达系统,已被广泛用作基因表达工具超过30年。
随着杆状病毒研究的进展,1983年首先发现杆状病毒可以被哺乳动物细胞内化,部分病毒DNA到达细胞核。但该部分DNA并没有持续存在,也不在哺乳动物的细胞中被转录(Tjia等,1983)。目前,重组杆状病毒已被证明能够进入除某些造血细胞系外的各种哺乳动物细胞,并在哺乳动物启动子的控制下表达外源基因。虽然脊椎动物不是杆状病毒的自然宿主,但由于其可以进入多种哺乳动物细胞,同时病毒DNA无法在哺乳动物细胞内复制或整合的特性,被应用于哺乳动物的基因传递。
杆状病毒外膜gp64糖蛋白是病毒包膜的主要组成部分,对病毒通过硫酸肝素受体介导的内吞作用进入细胞至关重要。在病毒进入后,gp64进一步诱导的内小体的染色体逃逸,从而将基因组DNA转运进入细胞质和细胞核。同样地,进入哺乳动物细胞后,病毒被转运到内小体,然后由gp64介导内小体逃逸,并在细胞质中诱导肌动蛋白丝的形成,促进病毒DNA转运到细胞核。其哺乳动物系统吧转导效率(21–90%)、转基因表达水平和持续时间(7–41天)。
鉴于高效的基因传递到多种细胞中,杆状病毒作为体内基因传递的载体已经引起了越来越多的兴趣。被靶向的组织包括兔颈动脉(Airenne等,2000)、大鼠肝(Huser等,2001)、Rat脑(Lehtolainen等,2002)、小鼠脑(Sarkis等人,2000)、小鼠骨骼肌(Pieroni等,2001)、小鼠大脑皮层和睾丸(tani等,2003)和鼠肝(Hoare等,2005)
间充质干细胞(MSCs)具有免疫特异性和免疫抑制性,能够自我更新和多向分化为多种细胞类型,包括脂肪细胞、软骨细胞和成骨细胞。由于这些特性,MSCs作为再生医学和基于MSC的细胞治疗产品的细胞来源已进入临床试验的各个阶段。其中由成人骨髓捐献者制成的同种异体MSC,正在进行3期临床试验。此外,可作为组织再生、癌症治疗、肾脏和心血管疾病治疗的基因工程细胞载体。与常见腺病毒、腺相关病毒(AAV)、逆转录病毒、慢病毒和质粒相比。杆状病毒能高效基因转移到关节软骨细胞(Hu,2004)、人骨髓间充质干细胞(Ho,2005)和骨髓间充质干细胞衍生祖细胞(Ho,2006)中。如将瞬时表达生长因子的杆状病毒工程化的MSCs植入骨髓间充质干细胞,可以促进骨修复,这将是治疗连接组织疾病,特别是软骨和骨疾病的候选细胞来源。
与上述基因递呈载体相比,至关重要的是杆状病毒转导MSC和MSC源性祖细胞的分化状态不受影响;未观察到转基因整合到宿主染色体和骨髓间充质干细胞核型的破坏;在杆状病毒载体转导的MSCs中,也未发现原癌基因上调或抑癌基因下调;转基因MSCs也没有诱导裸鼠肿瘤的形成。
目前杆状病毒在MSC上的两种应用可能:(1)MSCs可通过携带特定细胞因子的重组杆状病毒转导,通过分泌表达因子和旁分泌效应促进细胞(或调节)扩张或分化,省略在培养基中补充这些因子。(2)移植之前,杆状病毒可以在体外定向诱导MSC分化为目标祖细胞。细胞对杆状病毒的高易感性可提高细胞转化百分率,使目标基因在体内得到更高、更长的表达,加速组织再生或发挥治疗作用。
综上所述,杆状病毒可有效地转导入MSCs而不损害其扩增和分化能力,在体外环境下可能是一种有吸引力的替代转基因MSCs的方法。
AcMNPV抗病毒和肿瘤治疗的辅助手段:
接种杆状病毒可迅速建立非特异性抗病毒状态,动物实验发现接种AcMNPV可刺激NK,NKT细胞一小时内生产INF-γ,对抗致命病毒感染。如小鼠对抗脑心肌炎病毒(Gronowss基等,1999)或甲型流感病毒(Abe等,2003),鸡对抗感染性支气管炎病毒(Niu等,2008),及小鼠对抗口蹄疫病毒(Molina Guido Nicolás等,2020)。
AcMNPV激活DCs和NK细胞抗肿瘤:
AcMNPV还能刺激被感染细胞产生肿瘤坏死因子α、白细胞介素(IL)-1α和IL-1β,有效地刺激NK细胞介导的抗肿瘤免疫。研究发现,静脉注射AcMNPV感染的脾树突细胞和B细胞增加肝单核细胞的天然杀伤活性比例,血清干扰素(IFN)-γ水平升高。在肝转移模型中,AcMNPV分别诱导NKT细胞和IFN-γ抗肿瘤作用,使得用AcMNPV治疗的野生型小鼠的存活率显著提高;同时多次AcMNPV注射(第1、3和7天)引起类似的抗肿瘤作用,而且无动物毒性或致癌。因此,AcMNPV作为一种有效的抗肿瘤转移剂可能在临床试验中潜在地有用,并有望促进抗肿瘤治疗的发展。
杆状病毒具有优势:
无细胞毒性:杆状病毒转导对哺乳动物细胞无毒,即使在高MOI下也不会阻碍细胞生长,杆状病毒进行基因转导不会对哺乳动物造成任何可观察到的不良影响
安全性:杆状病毒在感染的哺乳动物细胞中不会复制,相对于其他具有复制能力病毒(RCV)基因治疗载体引起的严重安全问题,杆状病毒对人类没有致病性
多基因,容量大:杆状病毒的基因组较大(130kb),最大克隆能力至少为38kb,如此大的克隆能力为多种基因或较大的插入物提供了灵活性。与逆转录病毒和AAV载体相比,后者基因携带能力分别限制为7-7.5kb和3.5-4kb,并禁止调控序列或大基因片段的插入
病毒扩增容易,制备简单,生产成本低:与其它病毒形成鲜明相反的是,通过感染悬浮培养(如在旋转瓶或生物反应器)中并在感染后3-4天收获上清细胞,容易实现病毒的扩增。由于大规模昆虫细胞培养系统的完善,生产阶段只需在培养细胞中添加病毒溶液即可。此外,杆状病毒的构建、传播和处理可以在生物安全一级实验室中轻松地进行,而不需要专门的设备。
终上所述,杆状病毒具有插入多个基因的克隆能力大,细胞毒性作用最小,而且不在哺乳动物细胞中复制的优势,促使人们运用杆状病毒将外源基因传递到哺乳动物细胞中,使其成为新的细胞和基因治疗的载体。
MSC(Human Umbilical Cord Mesenchymal Stem Cells)人脐带间充质干细胞具有多种分化潜能,多功能干细胞在不同诱导条件培养基培养刺激下,表达多生物学活性,参与细胞增殖、分化、迁移(归槽)、免疫调节等功能。本发明扩增方法,通过筛选获得带有细胞因子等重组病毒导入MSC细胞中与NK细胞共培养,使NK细胞表达细胞表面激活受体(NKP46、DNAM-1、NKP30、NKP44、NKG2D)显著性增加,增强的NK细胞活性受体可有效地介导对靶细胞的溶解。(靶细胞为肿瘤及病毒感染的细胞)。
此外,MSC细胞与NK细胞共培养使NK细胞的分泌功能发生变化化,使NK细胞分泌多种细胞因子和趋化因子、穿孔素等,这些因子可刺激NK细胞介导靶细胞凋亡。
实验发现,伴随着MSC细胞与NK细胞共培养持续性激活,已激活的NK细胞对MSC细胞慢慢溶解。
MSC细胞与NK细胞共培养,NK细胞表达两种抑制受体与MHCI类分子相关,相比之下靶细胞缺乏MHCI类分子导致NK细胞活化去诱导细胞被杀伤。
MSC细胞诱导NK细胞表达还包括IL-18受体的炎症受体,结果表明NK细胞与MSC细胞共培养具备抗肿瘤能力。
本发明构建了含有细胞因子IL-2,IL-15,IL-21及CD抗体基因且带有哺乳动物CAG启动子的重组杆状病毒,用该重组杆状病毒呈递培养NK细胞必需细胞因子与CD抗体导入MSC细胞(人脐带间充质干细胞),刺激MSC细胞自然分泌人细胞因子与人CD抗体用于已被IL-2活化NK细胞共培养,促进NK细胞的增殖,改善NK细胞状态,提高NK细胞的抗病毒与抗肿瘤能力,培养完成后,收获NK细胞。整个程序满足GMP要求也符合临床使用标准。
本发明采用的组合性培养与诱导程序能有效地解决NK细胞产业化途中的如下三个重点技术瓶颈问题:
1、通过安全性强的病毒导入NK扩增的细胞因子与抗体,在NK细胞与MSC细胞共培养的条件下MSC细胞自然溶解成为滋养性细胞(Feeder cell),使NK的n天内扩增速增数千倍;
2、MSC细胞与NK细胞共培养有效地提高NK细胞杀伤靶细胞的活性,即通过激活NK细胞表面活性受体和分泌有效杀伤肿瘤细胞的炎性因子来增强NK细胞杀伤靶细胞的活性。
附图说明
图1a是pFBGFPR重组质粒;
图1b是pFB-HU-IL2重组质粒;
图1c是pFB-HU-IL15重组质粒;
图1d是pFB-HU-IL21重组质粒;
图1e是pFB-HU-IFN-γ重组质粒;
图2是重组杆状病毒中细胞因子基因递呈MSC细胞的验证试验结果;
图3是本发明扩增方法得到的NK细胞的杀伤活性测试结果;
图4是常规培养得到的NK细胞的杀伤活性测试结果;
图5a是本发明方法与常规培养方法制备的NK细胞上活性受体CD226的受体标记数结果;
图5b是本发明方法与常规培养方法制备的NK细胞上活性受体NKp46的受体标记数结果;
图5c是本发明方法与常规培养方法制备的NK细胞上活性受体NKp30的受体标记数结果;
图5d是本发明方法与常规培养方法制备的NK细胞上活性受体CD94的受体标记数结果;
图5e是本发明方法与常规培养方法制备的NK细胞上活性受体18Rα的受体标记数结果;
图5f是本发明方法与常规培养方法制备的NK细胞上活性受体18Rβ的受体标记数结果;
图5g是本发明方法与常规培养方法制备的NK细胞上活性受体CD3与CD56的受体标记数结果;
具体实施方式
下面结合具体实施例对本发明作进一步的说明,但并不局限于此。下述实施例中所述试剂原料除非注明来源外,均为市售的常见原料,试剂的配制采用常规方法。实施例中未详述的方法均为本领域常规操作。
实施例1:重组杆状病毒的构建
1、pFB-HU-IL2、pFB-HU-IL15、pFB-HU-IL21和pFB-HU-IFN-γ质粒载体的构建
1)分别在NCBI网站查询人IL-2基因序列、人IL-15基因序列、人IL-21基因序列和人IFN-γ基因序列序列;
2)利用PCR扩增法将得到人IL-2基因序列、人IL-15基因序列、人IL-21基因序列和人IFN-γ基因序列,备用;
3)将带有CMV启动子的pFBGFPR质粒(如图1a所示)进行AgeI-AccI双酶切,然后分别插入IL2,IL21,IL15和IFN-γ基因,获得以下重组质粒:pFB-HU-IL2(如图1b所示)、pFB-HU-IL15(如图1c所示)、pFB-HU-IL21(如图1d所示)和pFB-HU-IFN-γ(如图1e所示),
2、制备pFB-HU-IL2、pFB-HU-IL15、pFB-HU-IL21和pFB-HU-IFN-γ重组杆状病毒
1)将上述步骤获得的重组质粒载体转染至DH10Bac宿主菌中,1.5%LB琼脂糖培养板37度陪养过夜,挑取重组斑,37度扩增过夜;
2)提取重组杆状病毒的BacmidDNA,转染至sf9昆虫细胞中;
3)96小时后收取细胞培养上清,10000g离心获得上清为重组杆状病毒培养液;
4)将获得的重组杆状病毒1:500加入sf9昆虫细胞悬浮培养液中,培养96小时;
5)离心获得重组杆状病毒培养上清;
6)将获得的重组病毒超滤浓缩管离心浓缩10倍,4度保存,用于感染细胞实验。
通过上述步骤分别制备得到pFB-HU-IL2、pFB-HU-IL15、pFB-HU-IL21和pFB-HU-IFN-γ重组杆状病毒。
实施例2:间充质干细胞的制备
准备试剂:
a,间充质干细胞无血清培养基500ml/瓶2-8℃,避光保存(YDcon)
b,间充质干细胞无血清培养基添加剂5ml/瓶-20℃避光保存(YDcon)
c,0.05%胰酶-EDTA(sigma)
d,胰酶抑制剂500ml/瓶2-8℃避光保存(YDcon)
e,PBS x 20
间充质干细胞(MSC细胞)制备步骤如下:
1.从妇产科房收集健康脐带置存有25ml(150u庆大/PBS)的50ml无菌离心管中,4℃冷链至实验室。
(要求:取脐带时同时采脐带血,将脐血送艾迪康检测,或医院提供产妇血液报告。)
2、实验室留脐带组织少许留样-20℃冻存以备复查
用PBS(150u庆大/PBS)清洗脐带至无血色,沿脐带内腔纵向剪开脐带,用齿镊剥离脐静脉内膜,将覆盖动脉的华氏通(wharton)胶清洗剥离,撕开华氏通取出两根动脉。
3、用手术剪或手术刀将华氏通胶剪切成1mm3块大小均匀,用组织镊将脐带小块放置于贴壁培养皿,培养瓶底面,辅块密度为1块/cm2且用镊子使组织块均匀分布。
4、将对应体积的培养基沿培养皿或培养瓶瓶壁缓慢加入培养基必须没过脐带组织后缓慢移动,将培养皿或培养瓶放置37℃5%CO2,95%温度的培养箱中。
放置在二氧化碳培养箱中的培养皿或培养瓶,5天内(5x24小时)严禁移动。
5、自5天后,二天观察一次,培养液不足时应及时补液,从边缘小心缓慢添加。若有组织块周围已爬出细胞,且长至半径0.5cm,将组织块去除。
6、在显微镜下观察MSC细胞,当细胞融合度达到80%时,即可传代:弃去培养瓶(皿)中培养液,加入10mlPBS清洗细胞两次,加入2-4ml0.05%胰酶-EDTA消化细胞,显微镜下观察细胞变圆时立即臻臻加入10ml胰酶抑制剂,终止消化,收集细胞离心1000rpm/5分钟,沉淀加培养液慢慢混匀。按8000cell/cm2T75cm2=600000cell,加培液10-15ml。
重复6的步骤为P2。
细胞数量多则可用175cm2培养瓶以便传代。
扩增的干细胞p5加培养液.TGF.VC至80%融合率。
得到MSC细胞备用。
实施例3:活化NK细胞的制备
本实施例制备程序中培养基不含有动物蛋白质成分,含有重组或医疗级人类蛋白质的无血清细胞培养基,对人类外周血T淋巴细胞具有高增值性,适用于NK细胞的活化与扩增,是细胞免疫治疗用最佳培养基。
1、24孔培养板准备
(1)在24孔培养板中,加入Anti-CD16 MAb储备液。
(2)轻轻摇晃培养瓶,让溶液在培养培养瓶表面扩散。
(3)在室温下,孵育1小时或者保存在4℃直到使用前取出。移除包被溶液(MAb)。
(4)用生理盐水清洗培养瓶两次。洗过的培养瓶要立即使用。
2、血液分离
(1)将血液采集至含有抗凝剂(Heparin,ACD)的试管
(2)在15ml Lymphoprep上,小心地倒上20-30ml血液。请不要让血液和Lymphoprep混合在一起。
(3)室温下(大约20℃),用离心力800xg离心20分钟。如果血液储存超过2小时,将离心时间增至30分钟。
(4)离心后,血液被分为4层,由血浆(上层)、血浆和分离液之间的单核细胞(第2层)、Lymphoprep(第3层)和红细胞层(底层)构成。
3、制备热灭活人类血浆
(1)用无菌吸管采集上层的血浆,倒入灭菌过的离心管中,不要吸取到第2层的单核细胞。”
(2)56℃加热血浆30分钟。
(3)室温中,1200xg离心10分钟。
(4)用吸管将上清液采集至灭菌过的试管,保存在冰箱中,直到使用前取出。
4、制备
(1)用吸管将第2层单核细胞收集至灭菌过的离心管中。
(2)加入生理盐水稀释收集细胞,通过500xg离心10分钟沉淀细胞。
(3)倒弃或吸取以移除上清液。
(4)以生理盐水清洗细胞,500xg离心10分钟沉淀细胞。
(5)倒弃或吸取以移除上清液。
(6)重复(4)(5)一次
5、NK细胞增殖诱导
加入30mL含有IL-2(500IU/ml)ALyS505NK-AC培养基(ACTM),内含有5%灭活血浆,使细胞密度多于1x106cells/ml传代培养7天待用。
实施例4:重组杆状病毒中细胞因子基因递呈MSC细胞,再将携带细胞因子基因的MSC细胞与已激活NK细胞共培养
1、取一块24孔板,接种2.5×104/孔,实施例2制备的MSC细胞;
2、重组杆状病毒感染前将MSC细胞用1×PBS洗二遍;
3、步骤1中MSC细胞的24孔板每孔加实施例1步骤制备的重组杆状病毒加1ml(1.5×106/μl)加MSC无血清培养液0.5ml;
4、在25-27℃摇床上轻轻摇晃4小时;
5、补加MSC培养液1.5ml/孔,37℃、5%CO2培养箱中培养72h,得到IL2,IL21,IL15和IFN-γ导入的MSC细胞。上述制备得到的MSC细胞通过诱导表达,表达结果通过蛋白电泳后结果如图2所示,图2中:“M”为蛋白marker;
“CK1”为MSC细胞培养上清对照;
“CK2”为MSC细胞空白对照;
“1”为IFN-γ基因递呈的MSC细胞表达;
“2”和“3”为IL2基因递呈的MSC细胞表达;
“4”为IL15基因递呈的MSC细胞表达;
“5”IL21基因递呈的MSC细胞表达。
由此可见,重组杆状病毒中细胞因子基因均成功递呈MSC细胞。
6、吸去2mlMSC培养液/孔,MSC计数5×104/孔加入已培养7天的IL-2激活的NK细胞5×105,且MSC细胞与NK细胞的数量比为1:10,补加2ml NK细胞培养液/孔培养48小时,37℃5%CO2培养箱(此时MSC细胞已基本溶解);
7、NK计数2×106/孔,24孔板每孔传代10孔,每孔NK细胞起始浓度为2×105/孔,补加新鲜NK培液至2ml,培养36小时;
8、重复(循环)培养五次,计算NK细胞从0-25天扩增达5000培以上;
9、取样。作NK细胞杀伤活性测试、NK细胞表型及活性受体测定、以及NK细胞存活率鉴定。
检测方法1:NK细胞杀伤活性测试
试剂盒:CCK8试剂(KIT)
材料:10%小牛血清培养液,酶标仪,96孔平地板
操作步骤:
1、将肿瘤细胞株冻存管从-175℃液氮罐中取出置37℃水浴锅中化冰后倒入50ml离心管中,然后慢慢加入10%的小牛血清RPM了1640 1200rpm;
2、在96孔平地细胞培养板中,接种靶细胞(肿瘤细胞株A549),用10%小牛血清-RPM了1640培液稀释至细胞密度为2×105/ml,每孔加100μL;
3、在CO2培养箱培养2-4小时左右至肿瘤细胞贴壁后,弃去培养上清液;
4、分两组作为检测对象,具体如下:
第一组:按效靶比0:1(onlyA549),20:1,10:1,5:1,2.5:1,分为5例,靶细胞为肿瘤细胞株A549,效应细胞为实施例4中与MSC细胞共培养得到的NK细胞,其中每个效靶比均设置4个复孔;
第二组:按效靶比0:1(onlyA549),20:1,10:1,5:1,2.5:1,分为5例,靶细胞为肿瘤细胞株A549,效应细胞为常规培养的NK细胞(即未经MSC细胞共培养的NK细胞),其中每个效靶比均设置4个复孔;
还设有本底对照孔及悬浮NK细胞对照孔;
5、然后将96孔板置CO2培养箱中37℃培养过夜(约12个小时);
6、吸取上清液,用生理盐水漂洗三次,清除效应细胞及死亡肿瘤细胞,然后加入100μl 10%小牛血清RPM11640培液和10μl CCK8,37℃培养2-4小时;
7、在酶标仪450nm读取各效靶比光密度值/孔,结果如下:
第一组共培养组每个效靶比设置4个复孔,每个效靶比的平均值及本底如图3所示,具体数值如下:
效靶比0:1(onlyA549)为1.795;
效靶比20:1为0.442;
效靶比10:1为0.490;
效靶比5:1为0.836;
效靶比2.5:1为0.920;
本底对照为0.049;
悬浮NK细胞对照为0.051。
根据公式计算:杀伤率=1-[(ODET-ODE)/(ODT-ODC)]×100%。
注:T为靶细胞,E为效应细胞NK,ET为效应细胞作用于靶细胞,C为本底,即ODE=0.051,ODC=0.049,ODT=1.795;
通过计算得到:
效靶比20:1的杀伤率=1-[(0.442-0.051)/(1.795-0.049)]×100%=78%
效靶比10:1的杀伤率=1-[(0.490-0.051)/(1.795-0.049)]×100%=75%
效靶比5:1的杀伤率=1-[(0.836-0.051)/(1.795-0.049)]×100%=55%
效靶比2.5:1的杀伤率=1-[(0.920-0.051)/(1.795-0.049)]×100%=50%。
第二组非共培养组每孔效靶比设置4个复孔,每个效靶比的平均值及本底如图4所示,具体数值如下:
效靶比0:1(onlyA549)为1.792;
效靶比20:1为0.751;
效靶比10:1为0.797;
效靶比5:1为1.03;
效靶比2.5:1为1.12;
本底对照为0.049;
悬浮NK细胞对照为0.051。
根据公式计算:杀伤率=1-[(ODET-ODE)/(ODT-ODC)]×100%。
注:T为靶细胞,E为效应细胞NK,ET为效应细胞作用于靶细胞,C为本底,即ODE=0.051,ODC=0.049,ODT=1.792;
通过计算得到:
效靶比20:1的杀伤率=1-[(0.751-0.051)/(1.795-0.049)]×100%=40%
效靶比10:1的杀伤率=1-[(0.797-0.051)/(1.795-0.049)]×100%=43%
效靶比5:1的杀伤率=1-[(1.03-0.051)/(1.795-0.049)]×100%=56%
效靶比2.5:1的杀伤率=1-[(1.12-0.051)/(1.795-0.049)]×100%=61%。
上述结果可见:同样数值的NK细胞,本发明扩增方法制备的NK细胞与常规培养(非共培养)NK细胞,在效靶比20:1和10:1时,杀伤靶细胞的活性提高了近一倍。
检测方法2:NK细胞表型及活性受体测定
实验材料:(均购于美国Becton Dickinson公司)
萤光标记DNAM CD226,NKp46,NKp30,CD94,18Rα,18Rβ,CD3与CD56抗体操作步骤:
1、按照标记抗体使用说明,将适量抗制备成工作液;
2、取一定体积的细胞悬液于流式细胞测试管中,使细胞总数为1×106,1000rpm,5分钟;
3、弃上清液,重悬细胞,然后加入上述单抗工作液,充分混匀后,在4℃作用30分钟;
4、离心1500rpm,3min,弃上清;
5、用PBS洗涤细胞三次;
6、将细胞悬浮于0.5ml PBS中,流式细胞仪采样计数。
检测结果如下:
以下检测中本发明方法均是采用了MSC与NK细胞按照细胞数1:10的比例共培养的。
图5a是本发明方法与常规培养方法制备的NK细胞上活性受体DNAM CD226的受体标记数结果;
图5b是本发明方法与常规培养方法制备的NK细胞上活性受体NKp46的受体标记数结果;
图5c是本发明方法与常规培养方法制备的NK细胞上活性受体NKp30的受体标记数结果;
图5d是本发明方法与常规培养方法制备的NK细胞上活性受体CD94的受体标记数结果;
图5e是本发明方法与常规培养方法制备的NK细胞上活性受体18Rα的受体标记数结果;
图5f是本发明方法与常规培养方法制备的NK细胞上活性受体18Rβ的受体标记数结果;
图5g是本发明方法与常规培养方法制备的NK细胞上活性受体CD3与CD56的标记数结果;
从上述结果可以看到采用本发明方法制备的NK细胞的活性受体DNAM CD226+、NKp46+、18Rα+和18Rβ+的受体标记数均显著提升,提升了一倍左右,只有NKp30+活性受体略有下降,但是这不影响NK细胞的杀伤活性,而CD94+活性抑制受体显示有被抑制的趋势。
图5g显示本发明方法制备的NK细胞CD3-CD56+表型显著高于常规培养方法(即非共培养)得到的NK细胞,提升了约一倍左右。
由此可见,同样数值的NK细胞,本发明扩增方法制备得到的NK细胞与常规方法(即非共培养)得到的NK细胞在活性受体的激活上有显著性提升。
检测方法3:NK细胞存活率鉴定
PI(Propidium Iodide碘化丙啶)是一种细胞DNA特异荧光染料,死亡细胞或膜受损的细胞能染上PI荧光染料,而细胞膜完整的活性细胞对其拒染,应用流式细胞技术能区分死、活细胞,并计算出细胞成活率。
实验材料:
1、流式细胞仪
2、CELL Quest软件
3、PI(Propidium Iodide碘化丙啶):购于Sigma公司,工作液浓度为200ug/ml。
结果与计算:
以CELLQuest软件分析,在SSC、FSC图上,获取NK细胞群体,在点图上PI阳性细胞为死亡细胞,活细胞拒染,为阴性。计数10,000个细胞,测定PI阳性细胞的百分率并计算细胞存活率。细胞存活率=1-PI阳性细胞率%。
本发明扩增方法制备得到的NK细胞通过检测后结果如下:
阳性细胞数为360;
细胞存活率=1-360/10000*100%=96.4%。
以上实施例仅供说明本发明之用,而非对本发明的限制,有关技术领域的技术人员,在不脱离本发明的精神和范围的情况下,还可以作出各种变换或变型,因此所有等同的技术方案也应该属于本发明的范畴,应由各权利要求所限定。
Claims (6)
1.一种能显著增强杀伤活性的NK细胞扩增方法,其特征在于,包括以下步骤:
(1)构建含有NK扩增所需的细胞因子基因且带有哺乳动物启动子的重组杆状病毒;
(2)利用步骤(1)制备的重组杆状病毒对MSC细胞进行感染;
(3)将步骤(2)中感染后的MSC细胞加入已活化培养的NK细胞中共培养,共培养24-60小时后,NK细胞再继续传代扩增培养;
其中,所述步骤(1)中所述细胞因子为IL-2、IL-15、IL-21、IFN-γ中的至少一种。
2.根据权利要求1所述的能显著增强杀伤活性的NK细胞扩增方法,其特征在于,所述步骤(3)中,所述MSC细胞和NK细胞按照1:5-20的细胞数量比例混合共培养。
3.根据权利要求2所述的能显著增强杀伤活性的NK细胞扩增方法,其特征在于,所述步骤(3)中,所述MSC细胞和NK细胞按照1:8-12的细胞数量比例混合共培养。
4.根据权利要求1所述的能显著增强杀伤活性的NK细胞扩增方法,其特征在于,所述步骤(1)具体为:分别构建含IL-2、IL-15、IL-21和IFN-γ基因的质粒载体;分别用上述质粒载体制备带有哺乳动物启动子的重组杆状病毒。
5.根据权利要求1至4任一项所述的能显著增强杀伤活性的NK细胞扩增方法,其特征在于,所述步骤(1)中的哺乳动物启动子为CMV。
6.根据权利要求1所述的能显著增强杀伤活性的NK细胞扩增方法,其特征在于,所述步骤(3)中所述已活化培养的NK细胞是被IL-2活化的。
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