CN112746056A - 一种增强nk细胞毒作用的培养液及其制备方法 - Google Patents
一种增强nk细胞毒作用的培养液及其制备方法 Download PDFInfo
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- C12N2501/998—Proteins not provided for elsewhere
Abstract
本发明涉及细胞生物学技术领域,具体涉及一种增强NK细胞毒作用的培养液及其制备方法。一种增强NK细胞毒作用的培养液,所述无血培养液包括IL‑18、IL‑13、IL‑19、IL‑27、IL‑23、PRDX1、DEF1和1640基础培养基,IL‑18在培养液中的终浓度为5000‑8000IU/Ml,IL‑13在培养液中的终浓度为3000‑5000UI/ML,IL‑19在培养液中的终浓度为5000‑6000UI/ML,IL‑27在培养液中的终浓度为5000‑6000UI/ML,IL‑23在培养液中的终浓度为5000‑6000UI/ML,PRDX1的浓度为50μg/L、DEF1的浓度为60μg/L。本发明提供增强NK细胞毒作用的培养液与普通NK细胞培养液相比可明显增强NK细胞毒性及活性,为NK细胞在免疫细胞治疗中提供了广泛的应用。
Description
技术领域
本发明涉及细胞生物学技术领域,具体涉及一种增强NK细胞毒作用的培养液及其制备方法。
背景技术
NK细胞又称自然杀伤细胞(natural killer cell,NK细胞),是机体内重要的免疫细胞,作为与T细胞、B细胞并列的一大类淋巴细胞群体,是天然免疫系统的重要成员,负责杀伤老化、受病毒感染、肿瘤等异常细胞。
肿瘤是困扰人类健康的重大疾病,但由于其高度的复杂性、多样性、可变性和异质性,一直找不到一种更加深入治疗方法,随着医学的进步,癌症的治愈率、生存率都有显著的改善,但现在仍然是难治性的疾病。NK细胞的靶细胞主要是肿瘤细胞、病毒感染细胞、某些自身组织细胞(如血细胞)及寄生虫等,因此NK细胞是机体抗肿瘤、抗感染免疫的重要组成部分。由于这些特点,NK细胞在细胞免疫治疗上有着广泛的应用前景。在体外被活化的NK细胞毒性较高,可用作免疫细胞的治疗制剂。人体体外及动物试验证实了其对各种癌症治疗的可能性:利用肿瘤细胞系的方法,确认其对血液系统肿瘤(白血病和淋巴瘤等)、肝癌、肺癌、肾癌、神经系统肿瘤和皮肤癌等均具有体外细胞毒性,特别是对白血病和神经源性肿瘤已明确了其临床及非临床的治疗效果。2009年NK细胞治疗技术就已列入国家三类医疗技术目录。
充足的细胞数量和强烈的细胞毒性是细胞治疗疗效提升的关键因素。现有的细胞免疫治疗中的NK细胞主要是通过分离血液中的淋巴细胞,经体外培养、各种细胞因子刺激诱导扩增培养,得到一定量的细胞后回输人体,进行肿瘤治疗。现有NK细胞的培养基主要是由基础培养基、动物血清和各种因子培养组成,培养得到的NK细胞增殖倍数有限、数量不多,不能广泛的用于治疗,限制了NK细胞在免疫细胞治疗中的应用。目前急需一种细胞纯度高、扩增倍数大的NK细胞的的培养液及其培养方法。
发明内容
鉴于现有技术存在的问题,本发明的目的在于提供一种增强NK细胞毒作用的培养液及其制备方法。本发明提供的培养液与普通NK细胞培养液相比可明显增强NK细胞毒性及活性。
为实现上述发明目的,本发明采用以下技术方案。
一种增强NK细胞毒作用的培养液,所述无血培养液包括IL-18、IL-13、IL-19、IL-27、IL-23、PRDX1、DEF1和1640基础培养基,IL-18在培养液中的终浓度为5000-8000IU/Ml,IL-13在培养液中的终浓度为3000-5000UI/ML,IL-19在培养液中的终浓度为5000-6000UI/ML,IL-27在培养液中的终浓度为5000-6000UI/ML,IL-23在培养液中的终浓度为5000-6000UI/ML,PRDX1的浓度为50μg/L、DEF1的浓度为60μg/L。
所述的增强NK细胞毒作用的培养液的制备方法具体包括以下制备方法。
步骤1、基础培养基的制备。
1)溶解培养基:将干粉培养基10.4g溶于总量1/3的水中,再用水洗包装袋内面两次,倒入培养液中,振荡或超声助溶,一般不要加热助溶。
2)补加试剂:根据基础培养基说明和试验需要加入NaHCO3、谷氨酰胺。
3)加抗生素:一般抗生素终浓度为:青霉素100U/ml,链霉素100U/ml。市售青霉素为80万U/瓶,可溶于4ml体积,每一升培养液中加0.5ml即可。市售链霉素为100万U/瓶,可溶于5ml体积,每一升培养液中也加0.5ml即可。无链霉素的情况下,用庆大霉素代替,终浓度调为50~200U/ml。
4)调pH值:加水到800-1000ml,然后用5%NaHCO3调节pH到7.2。
5)过滤除菌:宜采用0.45um和0.22um滤膜各一张,上层为0.45um,下层为0.22um,以保证过滤效果。注意滤膜正面(光面)朝上。过滤后分装于小瓶中(100或200ml)。所得溶液为基础培养基。
步骤2、加小牛血清:根据培养基配制的量将小牛血清分装,-20℃冷冻保存。临用前将加入小牛血清(10%~20%体积分数)。
步骤3、在配制好的基础培养基中加入IL-18终浓度为7000IU/Ml、IL-13终浓度为4000UI/ML、IL-19终浓度为5500UI/ML、IL-27终浓度为5500UI/ML、IL-23终浓度为5500UI/ML、PRDX1(50μg/L)、DEF1(60μg/L)混匀后即可备用。
进一步地,所述的水为新制备的三蒸水,一般在试验前当天或前一天制备为好。
本发明提供的增强NK细胞毒作用的培养液中组成成分及其药理作用如下。
IL-18属于IL-1配体家族,结构与IL-1蛋白家族相似。人IL-18cDNA编码193个氨基酸,半胱氨酸天冬酶在N端将其水解为成熟的IL-18,发挥其生物学活性。其位于染色体11q22.2-22.3,由6个外显子和5个内含子组成,cDNA全长约1.1kb。IL-18是一种作用强大的前炎症细胞因子,最具特征的功能是调节细胞增生,分化及细胞外基质生成,故IL-18在糖尿病肾病(DN)的发生、发展中起重要作用。它是一个前炎症因子,可调节多种细胞发育及细胞因子分泌。研究表明,它是独特的依赖细胞因子周围环境而刺激Thl和Th2细胞反应的细胞因子。能促进外周单个核细胞产生IFN-γ、IL-2和粒细胞巨噬细胞集落刺激因子等细胞因子,增强NK细胞和Thl细胞的细胞毒作用,促进T细胞的增殖,并在Thl细胞分化和免疫反应中有促进和调节作用。在免疫调节、抗感染、抗肿瘤及慢性炎症性疾病发病过程中起着重要作用。
IL-13由TH2细胞产生,分子量约10KD。IL-13可诱导单核细胞分化,增强其MHCⅡ类分子的表达;抑制lps诱导的单核因子分泌,控制炎症反应;诱导B细胞增殖及合成IgE类抗体,增强B细胞表面MHCⅡ类分子、CD23及CD72的表达;协同IL-2刺激NK细胞产生IFN,从而促进单核-巨噬细胞活化和TH1型细胞免疫反应。IL-13还具有抑制HIV-1在巨噬细胞内复制,诱导中性粒细胞中IL-1RA基因表达和蛋白质合成等多种功能。
IL-19:成熟区153个氨基酸,与IL-10有同源性;染色体1q32。对抗原呈递细胞具有调节和促增殖效应。活化Stat3,受体为IL20R1/IL20R2。
IL-27属于IL-12/IL-6家族成员。主要由抗原呈递细胞产生,能促进T细胞增殖并向T辅助淋巴细胞(Thl)细胞方向分化,分泌γ干扰素IFN,在抗感染免疫及抗肿瘤免疫等方面发挥作用,参与机体多种疾病的演变过程。与IL-12有同源性,异源双聚体,α链为p28,与IL-12p35同源,其β链为EBI3。来源:IL-27由p28(IL-12的p35相关多肽)和EBI3(EB病毒诱导的基因3)组成异二聚体。其中p28与IL-12的p35亚基同源,EBI3与IL-12的p40亚基同源。人p28和EBI3基因分别定位于16p11和16p13.3。p28必须与EBI3结合,才具有IL-27生物学活性。由抗原呈递细胞活化早期阶段产生,促进naive T细胞增殖,与IL-12协同刺激T细胞的γ干扰素产生,促进早期Th1细胞。在T细胞增殖分化早期,可以促进CD4T细胞向Thl方向分化,同时IL-27可以协同IL-2促进初始T细胞产生IFN-γ;促进单核细胞产生IL-1、12、18和IFN等炎症细胞因子。它还可以抑制辅助性17T细胞和诱导型调节性T细胞的发育。研究还发现IL-27可以诱导小鼠B细胞产生LgG2a,抑制IL-4诱导的IgG1的合成;但IL-27可以诱导人B细胞产生LgG1。
IL-23与IL-12有同源性,异源双聚体,α链为p19,含189个氨基酸,与IL-12p35同源性,染色体12q13,其β链为IL-12的p40。经Stat4活化PHA刺激的T细胞,促进其增殖和γ干扰素产生,并诱导记忆性T细胞的增殖。
PRDX1过氧化氧化还原蛋白1(peroxiredoxins1,PRDX1)是新近发现的一种重要的细胞内抗氧化蛋白,PRDX1基因定位于人染色体1p34.1上,长度为15Kb,有四个转录子,都编码PRDX1蛋白。PRDX1参与细胞的氧化还原调节,主要通过硫氧还蛋白系统而不是通过谷氧还蛋白来降低过氧化物和还原当量,可能在减少过氧化物产生的新陈代谢过程中发挥重要作用。PRDX1可以通过硫氧还蛋白还原、清除过氧化物或超氧化物,在对抗ROS、细胞抗氧化防御过程中发挥关键作用,也可能通过调节细胞内H202的聚积来参与生长因子和肿瘤坏死因子的信号级联放大。研究表明,PRDX1作为细胞内有害活性氧的清除剂,对于肿瘤细胞具有双重作用:一方面PRDX1可阻碍正常细胞因活性氧导致DNA等损伤而向恶性转化,抑制肿瘤的发生;另一方面PRDX1也可抑制已转化的肿瘤细胞内大量活性氧诱导的凋亡,促进肿瘤细胞的存活,起到肿瘤支持与保护作用。
DEF1(degradation factor 1)是一种新的端粒调节蛋白,当酵母细胞DNA受到损伤,DNA转录依赖的DNA修复过程中RNApolymerase II的泛素化降解正是由Def1p来控制的,Def1p和y Ku蛋白一起协同的保护染色体末段端粒DNA不被降解。
与现有技术比,本发明具有以下有益效果。
本发明提供的一种增强NK细胞毒作用的培养液,所述无血培养液包括IL-18、IL-13、IL-19、IL-27、IL-23、PRDX1、DEF1和1640基础培养基,实验证明与普通NK细胞培养液相比可明显增强NK细胞毒性及活性。
附图说明
图1是15d收集NK细胞图(10×20)。
具体实施方式
为使得本发明的发明目的、特征、优点能够更加的明显和易懂,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,下面所描述的实施例仅仅是本发明一部分实施例,而非全部的实施例。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它实施例,都属于本发明保护的范围。
实施例。
一种增强NK细胞毒作用的培养液,所述无血培养液包括IL-18、IL-13、IL-19、IL-27、IL-23、PRDX1、DEF1和1640基础培养基,IL-18在培养液中的终浓度为5000-8000IU/Ml,IL-13在培养液中的终浓度为3000-5000UI/ML,IL-19在培养液中的终浓度为5000-6000UI/ML,IL-27在培养液中的终浓度为5000-6000UI/ML,IL-23在培养液中的终浓度为5000-6000UI/ML,PRDX1为50μg/L、DEF1为60μg/L。
本发明基础培养基(1640培养基)配方组成及浓度见表1。
表1.基础培养基(1640培养基)配方组成及浓度。
所述的增强NK细胞毒作用的培养液的制备方法具体包括以下制备方法。
步骤1、基础培养基的制备。
1)溶解培养基:将干粉培养基10.4g溶于总量1/3的水中,再用水洗包装袋内面两次,倒入培养液中,振荡或超声助溶,一般不要加热助溶。
2)补加试剂:根据基础培养基说明和试验需要加入NaHCO3、谷氨酰胺。
3)加抗生素:一般抗生素终浓度为:青霉素100U/ml,链霉素100U/ml。市售青霉素为80万U/瓶,可溶于4ml体积,每一升培养液中加0.5ml即可。市售链霉素为100万U/瓶,可溶于5ml体积,每一升培养液中也加0.5ml即可。无链霉素的情况下,用庆大霉素代替,终浓度调为50~200U/ml。
4)调pH值:加水到800-1000ml,然后用5%NaHCO3调节pH到7.2。
5)过滤除菌:宜采用0.45um和0.22um滤膜各一张,上层为0.45um,下层为0.22um,以保证过滤效果。注意滤膜正面(光面)朝上。过滤后分装于小瓶中(100或200ml)。
步骤2、加小牛血清:根据培养基配制的量将小牛血清分装,冷冻保存(-20℃)。临用前将加入小牛血清(10%~20%)。
步骤3、在配制好的1640培养基中加入IL-18终浓度为7000IU/Ml、IL-13终浓度为4000UI/ML、IL-19终浓度为5500UI/ML、IL-27终浓度为5500UI/ML、IL-23终浓度为5500UI/ML、PRDX1(50μg/L)、DEF1(60μg/L)混匀后即可备用。
进一步地,所述的水为新制备的三蒸水,一般在试验前当天或前一天制备为好。
本发明中制备培养液的器皿清洗要绝对干净,烤干后备用(滤器、滤膜、量筒、移液管及盛放培养基的小瓶等存放和转移培养基液体的器具都应进行灭菌)。
实施例。
一、培养液配方组成。
配方1:IL-18终浓度为(5000-8000IU/Ml)IL-13终浓度为3000-5000UI/ML、IL-19终浓度为5000-6000UI/ML、IL-27终浓度为5000-6000UI/ML、IL-23终浓度为5000-6000UI/ML、PRDX1(10μg)、DEF1(8.6μg)、基础培养基。
配方2:IL-18终浓度为(5000-8000IU/Ml)IL-13终浓度为3000-5000UI/ML、PRDX1(10μg)、DEF1(8.6μg)、基础培养基。
配方3:IL-19终浓度为5000-6000UI/ML、IL-27终浓度为5000-6000UI/ML、IL-23终浓度为5000-6000UI/ML、PRDX1(10μg)、DEF1(8.6μg)、基础培养基
配方4:PRDX1(10μg)、DEF1(8.6μg)、基础培养基。
配方5:IL-18终浓度为(5000-8000IU/Ml)IL-13终浓度为3000-5000UI/ML、IL-19终浓度为5000-6000UI/ML、IL-27终浓度为5000-6000UI/ML、IL-23终浓度为5000-6000UI/ML、基础培养基。
配方6:仅包括基础培养基。
配方1至配方6的培养液配方组成见表2。
表2.配方1至配方6的培养液配方组成。
2、NK细胞分离制备。
(1)NK细胞采自健康志愿献血者。按知情同意原则,所有研究对象均签署知情同意书。常规操作采集25例健康人外周血,避免样本污染。
(2)超净工作台中,分装血样于50L离心管中,取全血样1mL计数(3mL全血留样)。
(3)2000pm离心10min,取上层血浆于50mL离心管(1.5mL未灭活血浆留样)。56℃灭活30min,3000pm离心10min,去沉淀后4℃保存上清。
(4)生理盐水1:1稀释混匀血细胞,缓慢加到装有Ficoll的离心管内(界面清血样:Ficoll不超过2:1),1600rpm离心20min。
(5)吸弃上清,吸取白膜层到离心管,加生理盐水至45mL,混匀后1500pm离心10min。
(6)吸弃上清,生理盐水重悬后合并一管,加生理盐水至45mL。混匀,取样计数计算细胞总量,1800rpm离心10min。弃上清,计数。
(7)0d,T75培养瓶接种,每5人的NK细胞样本为一组,共分为7组,分别加入实施例1-6,第7组仅加入NK细胞基础培养基(每组的培养基中均含有血浆)CO2,37℃。(细胞密度为1.5x106-3x106)。
(8)3d,离心换液:T75培养瓶中细胞转移至50m1离心管1600rpm/min离心10min去除上清。细胞沉淀+30mL细胞培养液(含血浆)于新T75培养瓶。(每组加入对应的培养液)。
(9)5d,转移到T175培养瓶,细胞状态好,加培养液到50mL(含血浆)。
(10)8d观察细胞,细胞状态好,加培养液到120mL(含血浆)。
(11)11d每组细胞均补充50ml培养液(含血浆),继续培养。
(12)13d每组细胞均补充30ml培养液(含血浆),继续培养。
(13)15d收集细胞,如图1所示。
3、流式细胞术检测每组细胞表面标记物。
细胞培养15d后,流式检测细胞表面标记物,结果见表3,加入三种白细胞介素后,NK细胞活性增强。
表3.细胞培养15d后流式检测细胞表面标记物的结果。
CD3-CD56 | CD3+CD56 | |
实施例1 | 16.7 | 17.1 |
实施例2 | 10.4 | 13.8 |
实施例3 | 11.3 | 11.2 |
实施例4 | 10.4 | 8.7 |
实施例5 | 13.5 | 12.9 |
实施例4 | 5.1 | 7.7 |
4、CFSE/7-AAD双染法检测NK细胞毒作用。
(1)取培养13~14d的NK细胞及靶细胞,洗涤去除培养液,RPMI1640培养液调整NK细胞浓度为2x106,备用。
(2)CFSE/7-AAD法标记靶细胞:用DPBS液洗涤K562细胞2次,调整细胞数量为2×107个/ml。加入CFSE,终浓度为2.5μmol/L,混匀后置于37℃水浴箱中,避光孵育10min。取出细胞,用含有10%FBS的冷RPMI1640充分洗涤。RPMI1640完全培养液重悬细胞,调整靶细胞密度为5×105个/ml,备用。
(3)效-靶细胞共培养:将处理好的效应细胞与靶细胞按不同的效靶比(20∶1、10∶1)加入U形底96孔培养板中,在25℃下100g离心1min使细胞密切接触。5%CO2 37℃培养箱,共培养4h。设置仅NK细胞或仅靶细胞孔作为阴性对照,用于计算自然死亡率。
(4)CFSE/7-AAD法靶细胞死亡率检测:收集96孔细胞培养板中的细胞,PBS液洗涤细胞2次,重悬细胞,加入4μl 7-AAD标记死亡细胞,混匀,室温避光孵育20~25min。PBS液洗涤1次。根据FSC和SSC确定靶细胞群,对CFSE+细胞进行设门,分析该细胞群中CFSE+/7-AAD+双阳性细胞所占比率即为靶细胞死亡率,(靶细胞死亡率=杀伤后靶细胞死亡率-靶细胞自然死亡率),不同效靶比靶细胞死亡率结果见表4。
表4.不同效靶比靶细胞死亡率。
20:1(效:靶) | 10:1(效:靶) | |
实施例1 | 80.2% | 76.3% |
实施例2 | 77.5% | 77.1% |
实施例3 | 64.1% | 61.2% |
实施例4 | 66.5% | 60.8% |
实施例5 | 70.6% | 69.9% |
实施例6 | 44.6% | 41.3% |
Claims (4)
1.一种增强NK细胞毒作用的培养液,其特征在于,所述无血培养液包括IL-18、IL-13、IL-19、IL-27、IL-23、PRDX1、DEF1和1640基础培养基,IL-18的浓度为5000-8000IU/Ml,IL-13的浓度为3000-5000UI/ML,IL-19在培养液中的浓度为5000-6000UI/ML,IL-27的浓度为5000-6000UI/ML,IL-23的浓度为5000-6000UI/ML,PRDX1的浓度为50μg/L、DEF1的浓度为60μg/L。
2.如权利要求1所述的增强NK细胞毒作用的培养液的制备方法,其特征在于,包括以下制备方法:
步骤1、基础培养基的制备:
1) 溶解培养基:将干粉培养基10.4g溶于总量1/3的水中,再用水洗包装袋内面两次,倒入培养液中,振荡或超声助溶;
2)补加试剂:加入NaHCO3、谷氨酰胺;
3) 加抗生素:一般抗生素终浓度为:青霉素100U/ml,链霉素100U/ml;青霉素为80万U/瓶,溶于4ml体积,每一升培养液中加0.5ml即可;链霉素为100万U/瓶,溶于5ml体积,每一升培养液中也加0.5ml即可;无链霉素时,用庆大霉素代替,浓度为50~200U/ml;
4) 调pH值:加水到800 800-1000ml,用5% NaHCO3调节pH到7.2;
5) 过滤除菌:用0.45um和0.22um滤膜各一张,上层为0.45um,下层为0.22um,以保证过滤效果,滤膜正面朝上;过滤后分装于100或200ml小瓶中,所得溶液为基础培养基;
步骤2、加小牛血清:根据培养基配制的量加入小牛血清分装,-20℃冷冻保存;
步骤3、在配制好的基础培养基中加入IL-18、IL-13、IL-19、IL-27、IL-23、PRDX1、DEF1,混匀后即可备用。
3.如权利要求1所述的增强NK细胞毒作用的培养液的制备方法,其特征在于,所述的水为新制备的三蒸水,在试验前当天或前一天制备。
4.如权利要求1所述的增强NK细胞毒作用的培养液的制备方法,其特征在于,加入小牛血清的体积分数为10%~20%,临用前加入。
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