CN111826354A - 一种nk细胞及在肿瘤治疗方面的应用 - Google Patents
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Abstract
本发明公开了一种NK细胞及在肿瘤治疗方面的应用。高杀伤活性NK细胞的获得是利用NK细胞过继性***细胞的关键之一。本发明发现,miR‑124‑3p高表达的NK细胞具有更强的杀伤活性,对肿瘤细胞具有更强的杀伤力,因而可以用于制备成抗肿瘤细胞制剂。
Description
技术领域
本发明属于过继性细胞免疫治疗领域,具体涉及一种NK细胞及在肿瘤治疗方面的应用。
背景技术
NK细胞是机体主要的天然免疫细胞,无需抗原致敏,不依赖抗体,即可直接识别并清除体内异常细胞,如肿瘤细胞、细菌或病毒等微生物感染细胞,故在机体早期抗肿瘤和抗感染的免疫应答中发挥极为重要的作用。除了可直接杀死肿瘤细胞外,NK细胞活化后还可通过分泌炎性细胞因子和趋化因子,与树突状细胞协同促进效应T细胞募集到肿瘤微环境中发挥抗肿瘤作用,从而有效地诱发后续的特异性抗肿瘤免疫应答。
过继性细胞免疫治疗是将淋巴细胞经体外刺激培养后回输给肿瘤病人,直接杀伤肿瘤细胞或激发机体免疫反应来杀伤肿瘤细胞,进行肿瘤治疗的一种生物治疗方法。近年来,随着医学技术的发展,基于NK细胞的肿瘤过继性细胞免疫治疗应用越来越广泛。
高杀伤活性NK细胞的获得是利用NK细胞过继性***细胞的关键之一。
基因组学是对生物体所有基因进行集体表征、定量研究及不同基因组比较研究的一门交叉生物学学科。基因组学主要研究基因组的结构、功能、进化、定位和编辑等,以及它们对生物体的影响。通过比较基因组学可以发现疾病或功能差异的基因靶标。
在前期的一项高杀伤活性NK细胞和低杀伤活性NK细胞的比较基因组学研究中发现了多种差异表达的基因,其中包括多种miRNAs。目前,究竟哪些差异表达的miRNAs可以作为靶标调控NK细胞对肿瘤细胞的杀伤活性尚没有公布。
发明内容
本发明是为了克服现有技术的不足,提供一种NK细胞及在肿瘤治疗方面的应用。
技术方案如下:
一种高杀伤力的NK细胞,其为一种miR-124-3p高表达的NK细胞。
上述高杀伤力NK细胞在制备***的药物中的应用。
有益效果:
高杀伤活性NK细胞的获得是利用NK细胞过继性***细胞的关键之一。本发明发现,miR-124-3p高表达的NK细胞具有更强的杀伤活性,对肿瘤细胞具有更强的杀伤力,因而可以用于制备成抗肿瘤细胞制剂。
附图说明
图1为培养14d的NK细胞的流式鉴定图,从该图中可以看出,CD3-CD56+表型细胞(即NK细胞)的比例达到83.75%,NK细胞培养成功。
图2为各组NK细胞中miR-124-3p表达水平的RT-PCR测定结果,从该图中可以看出,与空白组相比,miR-124-3p组NK细胞中高表达miR-124-3p,转染对照组中miR-124-3p表达水平与空白组基本一致。
图3为各组NK细胞对HepG2肝癌细胞的杀伤力,从该图中可以看出,miR-124-3p组NK细胞对HepG2细胞的杀伤力显著高于空白组、转染对照组NK细胞,miR-124-3p组NK细胞对肿瘤细胞的杀伤力显著优于空白组和转染对照组。
具体实施方式
实施例1:
一、试验材料
淋巴细胞分离液购自Solarbio。
NK细胞培养基购自CellGro SCGM,胎牛血清购自Gibco。
CD3-PerCP-Cy5.5和CD56-FITC购自BD。
miR-124-3p mimics、miR-124-3p NC、RT-PCR引物购自广州锐博。
LipofectamineTM2000转染试剂盒购自Invitorgen。
二、试验方法
1、NK细胞的培养和鉴定
按常规方法取健康献血者外周抗凝血100mL,用淋巴细胞分离液分离单个核细胞,再用含500U/mLrhIL-2的NK细胞培养基对单个核细胞重悬制成细胞密度为2×105/mL的细胞悬浮液,接种于培养瓶中,在37℃、5%CO2条件下培养,每2~3d半量换液1次,并且在每次换液时调整细胞数量至5×104/mL,培养14d后,收集细胞。
流式鉴定:收集培养14d的细胞,PBS洗涤,按照常规的流式细胞检测法加入PerCP-Cy5.5标记的CD3和FITC标记的CD56抗体对细胞表面标志物进行检测。
2、miR-124-3p转染、RT-PCR鉴定和CD3-CD56+表型测定
收集培养14d的细胞,PBS洗涤,用含5%胎牛血清的NK细胞培养基重悬制成细胞悬浮液,以每孔1×105个细胞的密度接种于6孔板,分为空白组(不转染任何基因)、miR-124-3p组(转染miR-124-3p mimics)和转染对照组(转染miR-124-3p NC),铺板12h后按照LipofectamineTM2000转染试剂盒说明书进行转染操作,将待转染的miR-124-3p mimics及miR-124-3p NC与脂质体LipofectamineTM2000分别用NK细胞培养基稀释并按一定比例混合,室温作用30min后加入待转染细胞,置37℃、5%CO2培养8h后换液,继续48h后收集细胞。
RT-PCR鉴定转染效果:
Trizol法提取细胞总RNA,检测总RNA纯度,纯度符合要求后,RT-PCR试剂盒将RNA逆转录成cDNA,再PCR扩增cDNA,最后取适量PCR产物行琼脂糖凝胶电泳检测扩增产物。
miR-124-3p上游引物(SequenceNO.1):5’-CGGGTAGCAGGCTTCTGAGT-3’;
miR-124-3p下游引物(SequenceNO.2):5’-AAACCCCTCTCTGTCGGTAGCT-3’;
GAPDH上游引物(SequenceNO.3):5’-AATGGGCAGCCGTTAGGAAA-3’;
GAPDH下游引物(SequenceNO.4):5’-TGAAGGGGTCATTGATGGCA-3’。
CD3-CD56+表型测定:
收集的细胞用PBS洗涤,按照常规的流式细胞检测法加入PerCP-Cy5.5标记的CD3和FITC标记的CD56抗体对细胞表面标志物进行检测。
3、流式细胞法检测miR-124-3p高表达NK细胞的杀伤力
收集空白组(不转染任何基因)、miR-124-3p组(转染miR-124-3p mimics)和转染对照组(转染miR-124-3p NC)转染并继续培养48h的NK细胞,PBS洗涤,按照常规的流式细胞检测法加入APC-Anti-CD107a,检测CD107a的表达水平。
4、CCK-8法检测miR-124-3p高表达NK细胞对肿瘤细胞的杀伤力
收集空白组(不转染任何基因)、miR-124-3p组(转染miR-124-3p mimics)和转染对照组(转染miR-124-3p NC)转染并继续培养48h的NK细胞,PBS洗涤,用含10%胎牛血清的RPMI-1640培养基制成2×106/mL的细胞悬液,作为效应细胞;取对数生长期的HepG2肝癌细胞,用含10%胎牛血清的RPMI-1640培养基制成2×105/mL的细胞悬液,作为靶细胞。分别取效应细胞悬液和靶细胞悬液各100μL(效靶比10:1)接种于96孔培养板,同时设单独效应细胞孔和单独靶细胞孔,每组5个复孔,在37℃、5%CO2条件下培养8h,各孔加入20μL CCK8试剂,孵育4h后,在450nm波长酶标仪上测定各孔吸光值(OD),求其平均值,按照如下公式计算各组NK细胞对HepG2肝癌细胞的杀伤率。
杀伤率(%)=[1-(实验组OD值-单独效应细胞组OD值)/单独靶细胞OD值]×100%
5、统计学处理
采用SPSS17.0统计软件包处理数据,用均数±标准差表示,组间比较采用t检验,P<0.05代表差异具有统计学意义。
三、试验结果
1、NK细胞的培养和鉴定
流式鉴定结果显示,培养14d后CD3-CD56+表型细胞的比例达到83.75%,如图1所示。而培养前CD3-CD56+表型细胞的比例仅为6.62%。这表明NK细胞已经被成功培养。
2、miR-124-3p转染、RT-PCR鉴定和CD3-CD56+表型测定结果
RT-PCR结果如图2所示,与空白组相比,miR-124-3p组NK细胞中高表达miR-124-3p,转染对照组中miR-124-3p表达水平与空白组基本一致。
各组CD3-CD56+表型细胞的比例如表1所示,无显著差异。
表1各组CD3-CD56+表型细胞的比例
3、NK细胞杀伤力测定结果(流式细胞法)
各组NK细胞杀伤活性标志分子CD107a的表达水平如表2所示,该结果表明,miR-124-3p组NK细胞中CD107a的表达水平显著高于空白组、转染对照组NK细胞中CD107a的表达水平,miR-124-3p组NK细胞的杀伤力显著优于空白组、转染对照组NK细胞。
表2各组NK细胞杀伤活性标志分子CD107a的表达水平
4、NK细胞对肿瘤细胞杀伤力测定结果(CCK-8法)
各组NK细胞对HepG2肝癌细胞的杀伤力如表3和图3所示,该结果表明,miR-124-3p组NK细胞对HepG2细胞的杀伤力显著高于空白组、转染对照组NK细胞,miR-124-3p组NK细胞对肿瘤细胞的杀伤力显著优于空白组和转染对照组。
表3各组NK细胞对HepG2肝癌细胞的杀伤力
上述试验说明,miR-124-3p高表达的NK细胞具有更强的杀伤活性,对肿瘤细胞具有更强的杀伤力,因而可以用于制备成抗肿瘤细胞制剂。
以上实施例旨在具体介绍本发明的实质性内容,但不应将本发明的保护范围局限于以上具体的实施例。
序列表
<110> 周桂英
<120> 一种NK细胞及在肿瘤治疗方面的应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 1
cgggtagcag gcttctgagt 20
<210> 2
<211> 22
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 2
aaacccctct ctgtcggtag ct 22
<210> 3
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 3
aatgggcagc cgttaggaaa 20
<210> 4
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 4
tgaaggggtc attgatggca 20
Claims (2)
1.一种高杀伤力的NK细胞,其特征在于:其为一种miR-124-3p高表达的NK细胞。
2.权利要求1所述的高杀伤力NK细胞在制备***的药物中的应用。
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ANITHA SOMANCHI: "Membrane Bound IL21 Promotes Natural Killer Cell Expansion Through mIR 124-3p Mediated Regulation", 《THE TEXAS MEDICAL CENTER LIBRARY》 * |
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