丙型肝炎肝硬化CD56+T细胞和NK细胞数量及抗肿瘤活性

摘要

目的: 研究CD56⁺T细胞和NK细胞在丙型肝炎肝硬化时的数量及抗肿瘤细胞活性变化.

方法: 丙型肝炎肝硬化患者18例, 快速肝穿获得肝组织, 分离肝脏单个核细胞(MNC), 流式细胞仪分析CD56⁺T细胞及NK细胞数量, 肝脏MNC和外周血单个核细胞(PBMC)分别与IL-2混合培养, 检测IFN-γ含量及抗肿瘤细胞活性.

结果: 健康志愿者、慢性丙型肝炎患者、丙型肝炎肝硬化患者肝脏MNC中CD56⁺T细胞数量百分比分别是20.4%±6.2%, 11.2%±3.1%和5.0%±1.6%; NK细胞比例分别是31.1%±9.7%, 31.6%±8.3%和18.3%±5.4%; 肝脏MNC的IFN-γ产量分别是7.4±2.4, 3.2±1.8和1.9±0.5 μg/L; 抗肿瘤细胞的细胞毒性分别是61.1%±17.1%, 59.2%±14.6%, 26.7%±8.5%. 以上四组数据均以肝炎肝硬化患者变化最为显著(P<0.05).

结论: 丙型肝炎肝硬化时肝脏CD56⁺T细胞和NK细胞数量及抗肿瘤细胞活性分别下降.

关键词: 丙型肝炎; 肝硬化; CD56⁺T细胞; NK细胞; 细胞数量; 细胞活性

Numbers and activities of CD56⁺ T cells and natural killer cells in cirrhotic livers with hepatitis C

Rong-Shan Fan, De-Jun Yu, De-Rong Sun

Rong-Shan Fan, De-Rong Sun, Department of Internal Medicine, the Second Hospital of Daqing City, Daqing 163461, Heilongjiang Province, China

De-Jun Yu, Department of Laboratory, the Second Hospital of Daqing City, Daqing 163461, Heilongjiang Province, China

Correspondence to: Rong-Shan Fan, Department of Internal Medicine, the Second Hospital of Daqing City, Daqing 163461, Heilongjiang Province, China. rsfan519@163.com

Received: July 11, 2005
Revised: August 1, 2005
Accepted: August 11, 2005
Published online: June 28, 2006

Abstract

AIM: To study the numbers and anti-tumor activities of CD56⁺ T cells and natural killer (NK) cells in cirrhotic liver with hepatitis C (HC).

METHODS: Hepatic mononuclear cells (MNC) were isolated from liver specimens obtained from the patients (n = 16) with HC-induced cirrhosis by liver biopsy. In addition, the numbers of CD56⁺ T cells and natural killer cells were determined by flow cytometry. Liver MNC and peripheral blood mononuclear cells (PBMC) were co-cultured with the interleukin-2 (IL-2), respectively, and the production of interferon-g (IFN-g) and the antitumor activity were measured.

RESULTS: The percentages of CD56⁺ T cells among hepatic MNC in health individuals, HC and HC-induced cirrhosis patients were 20.4% ± 6.2%, 11.2% ± 3.1% and 5.0% ± 1.6%, respectively; the proportions of NK cells among liver MNC in the three groups were 31.1% ± 9.7%, 31.6% ± 8.3% and 18.3% ± 5.4%, respectively; the productions of IFN-g in the three groups were 7.4 ± 2.4, 3.2 ± 1.8 and 1.9 ± 0.5 mg/L, respectively; the anti-tumor activities hepatic MNC in the three groups were 61.1% ± 17.1%, 59.2% ± 14.6%, and 26.7% ± 8.5%, respectively. For the above four groups of parameters, the changes in HC-induced cirrhosis patients was the most significant (P < 0.05).

CONCLUSION: The numbers and anti-tumor activities of CD56⁺T cells and NK cells are decreased in cirrhotic livers with HC.

Key Words: Hepatitis C; Liver cirrhosis; CD56⁺ T cell; Natural killer cell; Cell number; Activity

0 引言

丙型肝炎多无症状, 据日本和美国对输血后丙型肝炎患者随访10-29年, 发现35.1%-51%的患者发展为肝硬化, 10.6%-23.4%发展为肝癌, 死于肝病的占15.3%[1]. CD56⁺T细胞和NK细胞在丙型肝炎肝细胞受损伤机制中有重要作用[2-3], CD56⁺T细胞和NK细胞在丙型肝炎肝硬化时的免疫监视作用还不清楚.

1 材料和方法

1.1 材料

1组健康志愿者12例; 2组病毒性肝炎丙型慢性患者16例; 3组丙型肝炎肝硬化患者18例. 诊断标准: 2000-09中华医学会传染与寄生虫病学分会、肝病学分会联合修订的病毒性肝炎防治方案. 并排除HAV、HBV、HDV、HEV等病毒感染, 无自身免疫性肝炎, 无饮酒及用过对肝脏有损伤性的药物史. 采用快速肝穿法, 获取肝组织; 通过静脉采集外周血. 用剪刀将肝组织标本剪成小碎块, 然后用胶原酶(0.5 g/L)和DNAse (0.1 mg/L)在37℃ 20 min, 200目不锈钢网过滤, RPMI 1640细胞培养液悬浮, 洗涤3次, 以含100 kU/L肝素的330 g/L Percoll溶液悬浮, 离心2 000 r/min 15 min, 室温. 然后放在红细胞溶解液中, 50 mL/L FBS-RPMI冲洗2次, 得MNC, 用淋巴细胞分离液将外周血标本离心得PBMC.

1.2 方法

肝脏MNC或PBMC用FITC标记的抗NKR-P¹抗体染色, PE标记的抗CD56抗体染色, 藻红蛋白-花青甘标记的抗αβTCR抗体染色, 用流式细胞仪分析, 0.1 L (0.01 mg/L)抗CD3抗体在4℃平底96孔细胞培养板培养过夜使抗体化, 培养前冲洗3次. 肝脏MNC和PBMC放在含有100 mL/L血浆的RPMI 1640液0.2 mL, 与固定的抗CD3抗体在96孔细胞培养板共同培养, 50 mL/L CO₂, 37℃. 肝脏MNC加入IL-2 (20 mg/L), 48 h后, 收集上清液储存在-80℃以备ELISA检测, 肝脏MNC培养5 d后, 做细胞毒性试验. ELISA kit法检测MNC培养上清液内IFN-γ水平, 按试剂盒说明书操作, 试剂盒由晶美生物工程有限公司提供. 对NK细胞敏感的YAC-1细胞为靶细胞, 放入含有100 mL/L FBS的RPMI 1640细胞培养液, 与3.7 MBq Na₂(⁵¹Cr)O₄共孵育60 min, 37℃, 肝脏MNC和PBMC为效应细胞, E/t = 10∶1, 孵育4 h后离心, 收集上清, Gamma计数器计数, 自然释放率<15%.

统计学处理 两组数据分析用t检验, 3组数据分析用方差分析.

2 结果

2.1 肝脏MNC中CD56⁺T和NK细胞

(1)肝脏MNC中的CD56⁺T细胞的比例在各组中依次是: 1组20.4%±6.2%, 2组11.2%±3.1%, 3组5.0%±1.6%(P<0.05). (2)肝脏MNC中的NK细胞的比例在各组依次是: 1组31.1%±9.7%, 2组31.6%±8.3%, 3组18.3%±5.4%(P<0.05). 丙型肝炎肝硬化时下降明显, 相反, CD56^-T细胞不下降. PBMC中这些细胞成分在各组无显著性差异.

2.2 肝脏MNC在IL-2刺激下产生IFN-γ肝脏MNC在IL-2刺激下产生IFN-γ量依次是: 1组7.4±2.4 μg/L, 2组3.2±1.8 μg/L, 3组1.9±0.5 μg/L (P<0.05). PBMC在IL-2刺激下产生IFN-γ量各组无显著差异.

2.3 肝脏MNC在IL-2刺激下抗肿瘤细胞毒性

在IL-2刺激下肝脏MNC对肿瘤细胞YAC-1细胞的细胞毒性依次是: 1组61.1%±17.1%, 2组59.2%±14.6%, 3组26.7%±8.5%(P<0.05). PBMC在IL-2刺激下产生的抗肿瘤细胞毒性, 各组无显著差异.

3 讨论

本项研究证明, 肝脏MNC具有产生IFN-γ的潜在能力, 也具有抗肿瘤细胞毒性, 肝脏MNC比PBMC有更强的产生IFN-γ和抗肿瘤细胞毒性. 更重要的是肝脏MNC中的CD56⁺T和NK细胞数量在肝炎和肝硬化时逐步下降, 与此相一致的是肝脏MNC产生IFN-γ量也逐渐减少, 肝脏MNC对肿瘤细胞毒性也逐步下降. 以往研究证实, CCl₄诱导的实验性肝硬化小鼠肝脏NKT细胞数量比正常减少, 肝脏MNC抗小鼠HCC细胞株的细胞毒性也降低[4]. 肝脏MNC紧密地黏附在肝脏实质细胞, 提示肝细胞对NKT细胞有基质样细胞作用[5]. 肝细胞能够表达IL-7 mRNA, IL-7是影响NKT发育的重要细胞因子[6]. 成年鼠肝脏中含有多源祖细胞, 可以分化成各种淋巴细胞[7], 人和鼠肝脏NKT和NK来自骨髓或肝脏, 肝硬化抑制了这些细胞的增殖[8-10].

IFN-γ在抗肿瘤免疫中有重要作用[11-12], 事实上IFN-γ受体缺失变异小鼠容易受到病毒感染[13-14]. 虽然CD56⁺T、CD56^-T和NK细胞在HCV抗原刺激时都产生IFN-γ, IL-2和IL-12, 但是在丙型肝炎及肝硬化时CD56^-T细胞数量不减少[15], 所以IFN-γ的减少可以认为是肝脏MNC中的CD56⁺T和NK细胞减少引起的.

最近有报道小鼠肝脏CD56⁺T和NK细胞在体外可以被IL-2或IL-12激活, 产生IFN-γ, 具有潜在的抗肿瘤活性, 抑制肿瘤转移和生成[16-17]. 人PBMC中CD56⁺T和NK细胞被IL-2或IL-12活化, 也可以获得抗肿瘤细胞活性[18]. 本项研究进一步证明人肝脏CD56⁺T和NK细胞在体外产生IFN-γ, 并且对肿瘤细胞有杀伤性, 有重要的抗肿瘤免疫作用, 而在丙型肝炎肝硬化时CD56⁺T和NK细胞的抗肿瘤作用减弱, 这可能是丙型肝炎肝硬化时易发生癌变的一个原因.

评论

背景资料

通过流行病学调查研究, 慢性丙型肝炎发展为肝硬化和肝癌的比例较高, 其确切机制目前还不清楚. 肝脏局部的免疫环境在肝癌的发生、发展中的作用越来越受到人们的重视. 肝脏中分布着大量天然和获得性的T细胞、NK细胞和CD56+T细胞(NKT), NKT同时表达T细胞受体和NK细胞受体, 是抗肿瘤早期起作用的效应细胞, 其重要性受到广泛的关注.

相关报道

Kawarabayashi et al研究了手术得到的标本, 分析了肝癌和非肝癌患者肝脏中NK和NKT细胞数量及抗肿瘤活性; Ogasawara et al认为IFN-g是NK及NKT发挥抗肿瘤作用的重要的细胞因子.

创新盘点

本文研究了丙型肝炎、肝硬化患者肝活检组织中的NK和NKT数量和活性, 从不同的角度证明NK和NKT在丙型肝炎肝硬化时数量减少及活性下降减弱了肝脏局部抗肿瘤免疫功能.

应用要点

改善肝脏局部的抗肿瘤免疫功能, 可能成为预防肝炎肝硬化发生肝癌的有效措施.

备注

电编:张敏 编辑:潘伯荣

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