CN104133058A - 用于检测nk细胞活性的试剂盒 - Google Patents
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Abstract
本发明提供了一种用于检测NK细胞活性的试剂盒。试剂盒包括PBS缓冲液、淋巴细胞分离液、RPMI1640培养液、荧光染料EGFP、抗体7-AAD-percp-cy5.5、荧光染料的载体pEGFP-N1质粒。本发明还提供了一种采用本试剂盒用于检测NK细胞的方法。本方法能节约检测时间和检测成本,且灵敏度高。
Description
技术领域
本发明型属于生物技术领域,涉及一种用于检测NK细胞活性的试剂盒。
背景技术
自然杀伤细胞(natural killer cell,NK)是机体重要的免疫细胞,不仅与抗肿瘤、抗病毒感染和免疫调节有关,而且在某些情况下参与超敏反应和自身免疫性疾病的发生,其发育成熟依赖于骨髓和胸腺微环境。NK细胞的功能主要是通过一下方式实现的。NK细胞自然杀伤活性:NK细胞识别靶细胞(某些肿瘤细胞、病毒感染细胞、某些自身组织细胞、寄生虫等),接着NK细胞释放穿孔素,细胞毒因子,活化的NK细胞释放TNF因子,从而达到裂解和杀伤靶细胞的可能。NK细胞毒作用(ADCC效应),主要是NK细胞表面具有FcγRⅢA,主要结合人IgG1和IgG3的Fc段(Cγ2、Cγ3功能区),在针对靶细胞特异性IgG抗体的介导下可杀伤相应靶细胞。活化的NK细胞可合成和分泌多种细胞因子,发挥调节免疫和造血作用以及直接杀伤靶细胞的作用。NK细胞通过自然杀伤和ADCC发挥的细胞毒作用,在机体抗病毒感染、免疫监视中起重要作用。
目前,NK细胞活性检测方法主要有形态法、酶释法、荧光法、放免法,这些诊断、检测方法的优缺点如下:形态法的方法简便,易于掌握,但判断死细胞与活细胞不免带有检测者的主观因素,也无法计数轻微损伤的细胞。酶释法的成本低、快速简便,可定量。缺点是靶细胞内碱性磷酸酶含量低或因某些未死亡细胞能自行释放,从而影响法灵敏度和特异性。此外乳酸脱氢酶分子较大,仅当靶细胞膜完全被破坏时才释放,故不能较早地反映效应的功能。荧光分析法常遇细胞自然释放率高,荧光本底强,影响灵敏度。放免法的操作简便快速能定量,缺点是自然释放率高,所需靶细胞数量多,半衰期短。
因此临床急需一种检测速度快且灵敏度高的检测技术,更有利于患者的及时诊治。
发明内容
为了解决上述技术问题,本发明的目的在于提供一种用于检测NK细胞活性的试剂盒,其能节约检测时间和检测成本,且灵敏度高。
为实现上述目的,在本发明的第一个方面,本发明提供了一种用于检测NK细胞活性的方法,包括步骤:(1)首先制备细胞:取3ml肝素钠抗凝的外周血与PBS缓冲液按照1:1稀释,将稀释后的标本缓慢注入具有2ml淋巴细胞分离液的10ml离心管中,以665g转速离心20min,吸取中间白细胞层;采用PBS清洗2次,然后以393g转速离心10min,去上清液;将500μl RPMI1640培养液加入离心得到的沉淀中,混匀,得到单个核细胞(PBMNC),浓度调节为1*106/ml备用;取对数期的K562-EGFP细胞用PBS洗2次,然后以393g转速离心10min,去上清液,加500μl RPMI1640培养液混匀,放室温静置1h,浓度调节为1*105/ml备用;(2)将100μl K562-EGFP加入1号管(对照管)中;将100μl PBMNC+100μl K562-EGFP加入2号管(检测管)中;然后将1号管与2号管均放入5%CO2培养箱中孵育2h;(3)将5.520μl抗体7-AAD-percp-cy5.5加入1号管与2号管中,充分混匀,静置1分钟,然后采用流式细胞仪检测细胞活性。
其中,在采用流式细胞仪检测细胞活性前需要调整其电压,调整电压的步骤为:将100μl PBMNC加入A管中;将100μl PBMNC与100μl K562-EGFP加入B管中,然后将A管与B管均放入5%CO2培养箱中孵育2h;将5.520μl7-AAD-percp-cy5.5加入A管中,充分混匀,静置1分钟。
其中,被测1号管与2号管中细胞的流速限制在200-800个/s,收集P1门内细胞至少10000个。
其中,采用流式细胞仪检测得到的结果中,对照管中Q2门内细胞代表K562-EGFP细胞在试验过程中自身凋亡的细胞数,Q2+Q4门内细胞代表K562-EGFP细胞总数,Q2/(Q2+Q4)代表自身K562-EGFP凋亡比例,采用A表示;检测管中Q2门代表被NK细胞杀伤及自身凋亡的K562-EGFP细胞总数,Q2/(Q2+Q4)代表总的K562-EGFP凋亡比例,采用B表示;NK细胞活性=100*(B-A)%。
其中,正常人NK细胞活性在3.9%至19.5%,当两者之差小于3.9%时,结果为NK细胞活性降低;当两者之差大于19.5%时,结果为NK细胞活性增强。
在本发明的第二个方面,本发明提供了一种用于检测NK细胞活性的试剂盒,其用于实施本发明的第一方面的方法,包括盒体、盒盖和设置在盒体内的衬垫,所述衬垫上设有小孔,装有PBS缓冲液的试剂管、装有淋巴细胞分离液的试剂管、装有RPMI1640培养液的试剂管、装有荧光染料EGFP的试剂管、装有抗体7-AAD-percp-cy5.5的试剂管、装有荧光染料的载体pEGFP-N1质粒的试剂管被设置在所述小孔内。
通过以上技术方案,本发明的有益效果如下:
利用流式细胞仪,设计抗体检测NK细胞活性,节约检测时间和检测成本;有助于噬血细胞综合征,感染、肿瘤自生免疫性疾病的诊断与治疗后监测。
附图说明
图1为采用本发明的试剂盒检测某患者的未被NK细胞杀伤的结果示意图;
图2为采用本发明的试剂盒检测某患者的被NK细胞杀伤的结果示意图。
具体实施方式
下面结合实施例对本发明的具体实施方式作进一步描述,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。
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。
NK细胞中含有大量的酶,当机体遇到靶细胞时,NK细胞能快速识别,并合成与分泌大量的细胞因子、酶、穿孔素等活性物质来裂解和杀伤靶细胞。本发明基于以上理论设EGFP-K562/PBMNC/7-AAD-percp-cy5.5组合检测方法,7-AAD-percp-cy5.5能让杀伤的靶细胞着色,利用CantoII检测被杀死的靶细胞。原理是用转染EGFP的K562细胞与BPMC按照1:10的比例在5%CO2培养箱中孵育2h后,在加入7-AAD-percp-cy5.5,充分混匀,稍置片刻上机检测,单细胞悬液在鞘液的包裹下形成单细胞液流并流经检测窗口;仪器产生的激光会激发抗体上标记的荧光素从而发出特定波长的光;仪器对不同波长的光进行分别检测,并转换成电信号;电信号经计算机系统处理后可进行分析。
仪器:CantoII流式细胞仪
试剂:7-AAD-percp-cy5.5购自美国BD公司,PBS缓冲液购自北京中杉金桥生物技术有限公司,改良型PBMI1640培养基购自北京创欣慧宇科技有限责任公司,pEGFP-N1质粒购自天津鼎国生物科技公司、K562来自北京大学人民医院,淋巴细胞分离液购自天津市灏洋生物制品有限公司。
具体的技术方案如下:
将100μl PBMNC加入A管中;将100μl PBMNC与100μl K562-EGFP加入B管中,然后将A管与B管均放入5%CO2培养箱中孵育2h;将5.520μl7-AAD-percp-cy5.5加入A管中,充分混匀,静置1分钟,然后采用A管与B管调整电压。
首先采用A管调节好FSC、SSC、Percp-cy5.5的电压,收集至少10000个细胞;将已经调好的FSC、SSC电压应用到B管以调节好EGFP通道电压,收集至少10000个细胞;将调节好的FSC、SSC、Percp-cy5.5、EGFP电压应用到如下所述的1号管(对照管)与2号管(检测管)中,收集至少10000个细胞。
1号管:对照管(K562-EGFP/7-AAD-percp-cy5.5)
2号管:检测管(PBMNC/K562-EGFP/7-AAD-percp-cy5.5)
1号管(对照管)的结果如图1所示,Q3门表示未转染上EGFP的K562细胞,Q2门内细胞代表K562-EGFP细胞在试验过程中自身凋亡的细胞数,Q2+Q4门内细胞代表K562-EGFP细胞总数,Q2/(Q2+Q4)代表自身K562-EGFP凋亡比例,采用A表示;2号管(检测管)的结果如图2所示,Q3门表示未转染上EGFP的K562细胞与PBMNC细胞,Q2门代表被NK细胞杀伤及自身凋亡的K562-EGFP细胞总数,Q2/(Q2+Q4)代表总的K562-EGFP凋亡比例,采用B表示;NK细胞杀伤的比例就等于两者之差。
结果判断:
NK细胞活性=100*(B-A)%
A(1号管)=Q2/(Q2+Q4)=151/(151+9170)=0.016
B(2号管)=Q2/(Q2+Q4)=246/(246+1508)=0.140
NK细胞活性=100*(0.14-0.016)%=100*0.124%=12.4%
图1与图2中的Q1表示杂质碎片与自然死亡的PBMNC细胞。正常人NK细胞活性在3.9%至19.5%,当两者之差小于3.9%时,结果为NK细胞活性减低;反之如果两者之差大于19.5%,则结果为NK细胞活性增强。
上面已经举例说明了本发明的实施例。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
Claims (6)
1.一种用于检测NK细胞活性的方法,包括步骤:
(1)取3ml肝素钠抗凝的外周血与PBS缓冲液按照1:1稀释,将稀释后的标本缓慢注入具有2ml淋巴细胞分离液的10ml离心管中,以665g转速离心20min,吸取中间白细胞层;采用PBS清洗2次,然后以393g转速离心10min,去上清液;将500μl RPMI1640培养液加入离心得到的沉淀中,混匀,得到单个核细胞PBMNC,浓度调节为1*106/ml备用;
取对数期的K562-EGFP细胞用PBS洗2次,然后以393g转速离心10min,去上清液,加500μl RPMI1640培养液混匀,放室温静置1h,浓度调节为1*105/ml备用;
(2)将100μl K562-EGFP加入对照管中;将100μl PBMNC+100μl K562-EGFP加入检测管中;然后将对照管与检测管均放入5%CO2培养箱中孵育2h;
(3)将5.520μl抗体7-AAD-percp-cy5.5加入对照管与检测管中,充分混匀,静置1分钟,然后采用流式细胞仪检测细胞活性。
2.根据权利要求1所述的一种用于检测NK细胞活性的方法,其特征在于,在采用流式细胞仪检测细胞活性前需要调整其电压,调整电压的步骤为:将100μl PBMNC加入A管中;将100μl PBMNC与100μl K562-EGFP加入B管中,然后将A管与B管均放入5%CO2培养箱中孵育2h;将5.520μl7-AAD-percp-cy5.5加入A管中,充分混匀,静置1分钟。
3.根据权利要求1所述的一种用于检测NK细胞活性的方法,其特征在于,对照管与检测管中细胞的流速限制在200-800个/s,收集P1门内细胞至少10000个。
4.根据权利要求1至3中的一个所述的一种用于检测NK细胞活性的方法,其特征在于,在采用流式细胞仪检测得到的结果中,对照管中Q2门内细胞代表K562-EGFP细胞在试验过程中自身凋亡的细胞数,Q2+Q4门内细胞代表K562-EGFP细胞总数,Q2/(Q2+Q4)代表自身K562-EGFP凋亡比例,采用A表示;检测管中Q2门代表被NK细胞杀伤及自身凋亡的K562-EGFP细胞总数,Q2/(Q2+Q4)代表总的K562-EGFP凋亡比例,采用B表示;
检测出的NK细胞活性=100*(B-A)%。
5.根据权利要求4所述的一种用于检测NK细胞活性的方法,其特征在于,正常人NK细胞活性在3.9%至19.5%,当检测出的NK细胞活性与正常人NK细胞活性两者之差小于3.9%时,检测结果为NK细胞活性降低;当两者之差大于19.5%时,检测结果为NK细胞活性增强。
6.一种用于检测NK细胞活性的试剂盒,其用于实施权利要求1-5中任一项所述的方法,所述试剂盒包括盒体、盒盖和设置在盒体内的衬垫,所述衬垫上设有小孔,装有PBS缓冲液的试剂管、装有淋巴细胞分离液的试剂管、装有RPMI1640培养液的试剂管、装有荧光染料EGFP的试剂管、装有抗体7-AAD-percp-cy5.5的试剂管、装有荧光染料的载体pEGFP-N1质粒的试剂管被设置在所述小孔内。
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