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⊙研究原著⊙
新型免疫活性细胞CIK体外对肝癌细胞的杀伤
杜清友1 王福生1 徐东平1 刘 洪1 雷周云1 刘明旭1 王业东1 陈菊梅1 吴祖泽2
1中国人民解放军302医院生物工程研究室
北京市
100039
2中国人民解放军军事医学科学院放射医学研究所
北京市
100850
项目负责人 王福生,
100039, 北京市丰台路26号，中国人民解放军302医院生物工程研究室.
收稿日期 2000-04-03
接收日期
2000-04-13
Cytotoxic effects of CIK against hepatocellular carcinoma cells in vitro
Qing You Du1, Fu Sheng Wang1, Dong Ping Xu1,
Hong Liu1, Zhou Yun Lei1, Ming Xun Liu1,
Ye Dong Wang1, Ju Mei Chen1 and Zu
Ze Wu2
1Division of Bioengineering,
the 302 Hospital of PLA, Beijing 100039, China
2Institute of Radiation
Medicine, Academy of Military Medical Sciences, Beijing 100850, China
Correspondence to Prof.
Fu Sheng Wang, Division of Bioengineering, Chinese PLA 302 Hospital,
26 Fengtai Road, Beijing 100039, China
Tel. 0086-10-66933332， Fax. 0086-10-63831870
Email. fswang@public.bta.net.cn
Abstract
AIM To
evaluate the proliferation profile and the antitumor
METHODS Peripheral
blood mononuclear cells(PBMC) were obtained
RESULTS CIK
cells increased significantly after 14 days of culture and expanded more than 500-fold in the medium
consisting of 100mL·
CONCLUSION CIK
cells were highly efficient cytotoxic effector cells against hepatocellular carcinoma cells and may
be used for adoptive antitumor immunotherapy.
Subject headings liver
neoplasms; immunocompetence; cytokine-induced killer cells; peripheral blood mononuclear
cells; lymphokine-activated killer; tumor-infiltrating lymphocytes
Du QY, Wang FS, Xu DP, Liu H, Lei ZY, Liu MX, Wang YD, Chen JM, Wu ZZ. Cytotoxic effects of CIK against
hepatocellular carcinoma
cells in vitro. Shijie Huaren Xiaohua Zazhi,
2000;8(8):863-866
摘要
目的
观察人体CIK(cytokine-induced
killer)细胞的体外增殖力及杀伤肝癌细胞活性.
方法
采取健康自愿者的成分血，用淋巴细胞分离液分离获得外周血单个核细胞（PBMC），在体外培养条件下，通过加入不同细胞因子（如IFN-γ,
IL-1, IL-2，CD3 mAb）培养诱导成CIK细胞和LAK细胞，观察不同培养细胞的增殖能力；用流式细胞仪对培养细胞做细胞表型动态分析，观察培养细胞的表面标志；与LAK细胞作对比，用MTT法测定并比较不同效应细胞的体外抗肝癌细胞活性.
结果
CIK细胞在培养14d后开始大量增殖且在含人血清的培养基
结论
CIK细胞是一种具有较强杀伤肝癌细胞的免疫活性细胞，有可能应用于抗肿瘤的生物治疗.
主题词
肝肿瘤；免疫活性；CIK
细胞；外周血单个核细胞；淋巴因子激活的杀伤细胞；肿瘤浸润淋巴细胞
杜清友, 王福生, 徐东平, 刘洪, 雷周云, 刘明旭, 王业东, 陈菊梅, 吴祖泽.
新型免疫活性细胞CIK体外对肝癌细胞的杀伤.
世界华人消化杂志，2000；8(8)：863-866
0 引言
CIK细胞(cytokine-induced killer)是一种有效杀伤白血病细胞的免疫活性细胞［1]. 在体外条件下，人体外周血单个核细胞(peripheral blood mononuclear
cells, PBMC)经过多种细胞因子(如IFN-γ, IL-1, IL-2,
anti-CD3 mAb)的共同刺激后诱
1 材料和方法
1.1 材料 基因重组人IL-2，IFN-γ和CD3 mAb购自北京邦定生物医学公司；基因重组人IL-1α及
1.2 方法
在Cobe Spectra型采血机上(301医院血库)用淋巴细胞采集程序采集健康自愿者成分血，所得样本体积为50mL，用等体积淋巴细胞分离液纯化所得成分血，获取PBMC.
1.2.1 CIK细胞［18,19］
用1640完全培养液(含100mL·L-1小牛血清或人AB型血清，50mg·L-1氨苄青霉素，10mmol·L-1
Hepes, 50μmol·L-1
2-巯基乙醇)或无血清培养液将PBMC调至1.5×109·L-1mL，d0加入IFN-γ
1×106U·L-1, 37℃，50mL·L-1 CO2孵箱中培养24h后，加入IL-1α
1×105U·L-1, IL-2
3×105U·L-1,
CD3 mAb 50μg·L-1
1.2.2 LAK细胞［20,21］
将PBMC调至2×109
1.2.3 体外细胞毒活性测定［22,23] 以不同效靶比(2.5∶1, 5∶1, 10∶1, 20∶1)将CIK或LAK效应细胞与7402或HepG2肝癌细胞一起放入96孔培养板中，另设单独靶细胞和单独效应细胞对照孔，每组设3孔，37℃，50mL·L-1 CO2孵箱中培养4h，用MTT显色法测定.
杀伤活性(%)=［1-(实验组A值-单独效应细胞A值)÷单独靶细胞A值］×100%
2 结果
2.1 效应细胞的增殖与表型分析
首先，采血机采集成分血后，用淋巴细胞分离液可一次获得足够数量的PBMC.
用含100mL·L-1牛血清的1640完全培养基（含细胞因子）来培养细胞.
实验发现，培养细胞在4d～5d出现增殖，快速增长期在14d～28d之间，至28d时，细胞数量达到最大值，随后细胞数量开始减少. CIK细胞最大增殖倍数可达400多；而培养的LAK细胞虽有一定的增殖，但其最大增殖倍数仅为14，远远低于CIK细胞的增殖倍数(P＜0.001,
T检验，图1).
不同培养时间的CIK细胞经流式细胞仪表型分析表明，CIK细胞属于异质细胞群. 在培养过程中CD3+CD56+双阳性细胞的百分含量大幅度上升，由起初的0.13%可以上升到21d的19.5%，而到56d时可达到51.26%. 由此可见,
CD3+CD56+细胞的绝对数量得到了大量的增殖(图2).
图1 效应细胞的增殖数量与培养时间的关系.
图2 CD3+CD56+细胞含量与培养时间的关系.
2.2 不同培养基对CIK增殖的影响
分别用含牛血清、人AB血清的培养基及无血清培养基，来观察不同培养基对CIK细胞增殖的影响. 实验发现，CIK细胞在含人AB血清的RPMI1640培养基中增殖最好，在培养至28d时，到达其最大增殖倍数500多，而无血清培养基中CIK细胞开始增殖速度较快（7d～14d），但随后增殖缓慢（14d以后），其最大增殖倍数仅约为60(图3).
图3 不同培养基中CIK与培养时间的关系.
2.3 体外杀伤肝癌细胞活性
利用MTT检测法，通过体外对肝癌细胞7402和HepG2的杀伤实验表明，CIK细胞在培养2wk～3wk时杀伤肝癌细胞活性最高；而LAK细胞则在培养7d～10d时体外杀伤肝癌细胞活性最高，CIK细胞的杀伤活性显著高于LAK细胞(图4).
3 讨论
CIK细胞是一类非主要组织相容性复合物（major histocompatibility complex,
MHC）和非T细胞受体(T cell receptor, TCR)限制性的免疫活性细胞［24,25］，其主要效应细胞为CD3+CD56+. 在培养过程中，一些非活性细胞在多种细胞因子的共同刺激下，激活为有细胞毒作用的CIK细胞［26,27］.
这些活性细胞发挥抗肿瘤作用的机制很可能是通过粘附因子LFA/ICAM-1途径与肿瘤细胞相互结合后，能够分泌含有大量BLT脂酶(benzyloxycarbonyl-1 lysine thiobenzyl ester, BLT
esterase)的细胞质颗粒，这些颗粒能够直接穿透封闭的靶细胞膜进行胞吐，从而导致肿瘤细胞的裂解［28］.
同时，CIK细胞自身还能分泌IL-2, IL-6, TNF-α和GM-CSF等一些细胞因子，提高细胞毒作用［29］，或者调节肿瘤细胞对CIK细胞的敏感性［30］.
成功地进行过继免疫治疗的关键问题是能否获得足够的并且具有较强活性的免疫活性细胞［31-33］. 而CIK细胞能够高效杀伤肿瘤细胞，且具有易于体外培养增殖、毒副作用小等优点. 这样应用CIK细胞治疗克服了过去过继免疫疗法的一些缺点如体外增殖数量少、需输注IL-2、低效及副作用大等，因而被认为具有良好的临床应用前景［34,35］. 我们通过改善CIK细胞的培养条件如培养基的类型、pH值的调整以及培养液的补加方式等，可使CIK细胞的最大增殖倍数达500多倍，接近国外报道的倍数(600倍)［36］，而远远高于国内所报道的增殖倍数［24］，其主要效应细胞CD3+CD56+双阳性细胞的绝对数量获得了大量增加，并且能够长期保持活跃的生长趋势(2wk～3wk).
肝癌在我国是一种多发性肿瘤，且肝癌患者大多数合并严重肝硬变［37,38］，易发生肝内播散和远处转移，严重危害着人们的生命和健康［39,40］.
目前，肝癌的治疗一般采用手术、化疗及放疗手段［41-45］，但患者经过上述治疗后，难以长期改变患者的生存质量，而且预后极差［46-48］.
因此，人们愈来愈重视依赖自身机体的抗癌细胞来发挥重要作用. 通过比较CIK和LAK细胞的生物学特征及其体外的抗肝癌细胞作用，结果表明CIK的抗肿瘤细胞活性显著优于LAK细胞，能够更好地满足临床上有效治疗所需的细胞数量和细胞毒活性［49,50］.
此外,
颐腔构鄄炝薈IK细胞在无血清培养基中的增殖情况. 实验发现，在所用的几种培养基中，培养至14d之前，无血清培养基中CIK细胞的增殖数量最高，但随后细胞增殖缓慢，细胞数量逐渐减少，其保持增殖能力时间较短，且其最大增殖倍数（60）远远低于含人AB血清培养基的（500），这很可能是由于无血清培养基本身所造成的. 这为以后CIK
细胞应用于临床治疗提供了实验依据.
总之，我们通过实验证实了在体外条件下CIK细胞具有增殖数量多，能够保持较长时间的高细胞毒活性，且体外杀伤肝癌细胞活性强等优点，是否在动物及人体内同样具有较强的抗肿瘤活性，这一问题正在进一步研究中.
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