拉米夫定体外对慢性乙型肝炎患者树突状细胞功能的影响

摘要

目的: 体外研究不同浓度拉米夫定(lamivu-dine, LAM)对慢性乙型肝炎(chronic hepatitis B, CHB)患者树突状细胞(dendritic cell, DC)功能影响.

方法: 分离慢乙肝患者外周血单核细胞, 在含GM-CSF+IL-4及不同浓度LAM (0, 0.125, 0.25, 0.5, 1, 2 mmol/L)培养条件下制备DC. 观察DC形态学变化并用MTT法测定DC刺激同种异体淋巴细胞增殖能力, 流式细胞仪(FCM)测定其细胞表型分子CD1a, CD83, CD80及HLA-DR的表达, ELISA法检测培养上清中IL-12和IL-6含量.

结果: 在LAM 0.5 mmol/L组, DC表型分子CD83, CD1a, HLA-DR的表达最高, CD80与LAM未处理组相比无明显差异. 与LAM未处理组相比, LAM 0.5 mmol/L处理组DC膜表面分子CD1a, CD83, HLA-DR表达增高(CD1a: 54.0±9.2 vs 33.6±10.1, P<0.05; CD83: 20.3±6.1 vs 11.8±6.2, P<0.05; HLA-DR: 74.5±7.1 vs 52.9±7.7, P<0.05); 其上清液中IL-12分泌水平增高(810.0±91.5 ng/L vs 268.0±34.3 ng/L, P<0.05), IL-6则显著降低(28.1±2.6 ng/L vs 55.3±7.4 ng/L, P<0.05); 刺激同种异体淋巴细胞增殖能力(SI)增强(1.9±0.6 vs 1.2±0.5, P<0.05).

结论: LAM体外可增强慢乙肝患者树突状细胞活性.

关键词: 慢性乙型肝炎; 树突状细胞; 抗原提呈; 拉米夫定

In vitro effects of lamivudine on function of dendritic cells derived from patients with chronic hepatitis B infection

Dong-Yun Zhang, Peng-Yuan Zheng, Xiao-Qin Zhang, Guang-Hui Liu, Hui-Ping Lou, Fu-Ai Tang, Jing-Xiu Bai, Yuan-Ming Qi

Dong-Yun Zhang, Peng-Yuan Zheng, Xiao-Qin Zhang, Guang-Hui Liu, Hui-Ping Lou, Fu-Ai Tang, Jing-Xiu Bai, Yuan-Ming Qi, Department of Digestive Disease, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450014, Henan Province, China

Supported by: the Medical Science and Technology Innovation Talent Project of Henan Province, No. 2004-45.

Correspondence to: Dr. Peng-Yuan Zheng, The Department of Digestive Disease, The Second Affiliated Hospital of Zhengzhou University, 450014, 2 JingBa Rd, Zhengzhou, China. medp7123@yahoo.com

Received: April 1, 2006
Revised: April 15, 2006
Accepted: April 20, 2006
Published online: June 18, 2006

Abstract

AIM: To investigate the effects of lamivudine on the function of dendritic cells derived from patients with chronic hepatitis B (CHB) in vitro.

METHODS: Dentritic cells (DCs) derived from the monocytes of CHB patients were cultured with interleukin-4 (IL-4) plus granulocyte-macrophage colony-stimulating factor (GM-CSF). Three days after being cultured, the cells were divided group A and B. Group A was treated with different concentrations of lamivudine (0, 0.125, 0.25, 0.5, 1, 2 mmol/L), while group B served as the control. Cell morphology was observed under light microscope and cell surface molecules including HLA-DR, CD80, CD83, and CD1a were assayed by flow cytometry. The concentrations of IL-6 and IL-12 in the supernatant were measured by enzyme-linked immunosorbent assay (ELISA). T cell proliferation induced by DC was detected by methyl thiazolyl tetrazolium (MTT).

RESULTS: The cells treated with 0.5 mmol/L lamivudine had the highest expression of CD1a, CD83 and HLA-DR, and the expression of CD80 was not significantly different between the cells with and without lamivudine treatment. After treatment with 0.5 mmol/L lamivudine, the expression of CD1a, CD83 and HLA-DR were significantly higher than those of DCs without lamivudine treatment (CD1a: 54.0 ± 9.2 vs 33.6 ± 10.1, P < 0.05; CD83: 20.3 ± 6.1 vs 11.8 ± 6.2, P < 0.05; HLA-DR: 74.5 ± 7.1 vs 52.9 ± 7.7, P < 0.05); the secretion of IL-12 was significantly increased in comparison with that of the control group (810.0 ± 91.5 ng/L vs 268.0 ± 34.3 ng/L, P < 0.05), while the level of IL-6 was lowered markedly (28.1 ± 2.6 ng/L vs 55.3 ± 7.4 ng/L, P < 0. 05); the stimulatory capacity of DCs in the allogeneic mixed leukocyte reaction was markedly enhanced as compared with that of the control group (stimulatory index: 1.9 ± 0.6 vs 1.2 ± 0.5, P < 0.05).

CONCLUSION: Pulsed with lamivudine in certain concentration, the biological activity of DCs derived from CHB patients can be efficiently enhanced.

Key Words: Chronic hepatitis B; Dendritic cells; Antigen presentation; Lamivudine

0 引言

慢性乙型肝炎(chronic hepatitis B, CHB)造成免疫耐受的重要原因之一是患者自身树突状细胞(dendritic cell, DC)功能低下, 抗原提呈功能缺陷, 不能有效地把病毒抗原的信号传递给机体免疫系统[1-2]. DC是唯一能激活初始化T淋巴细胞的抗原提呈细胞(antigen-presenting cell, APC), 在抗病毒的细胞免疫治疗中起重要作用[3-4]. 如何加强DC的抗原提呈能力, 发挥其免疫功能已成为治疗CHB急需解决的问题之一. 拉米夫定(lamivudine, LAM)为一口服核苷类药物, 在细胞内经磷酸化后, 与脱氧胞嘧啶核苷(dCTP)竞争进入病毒合成中的DNA链, 终止病毒DNA的复制[5-7]. 国外研究发现LAM在抗病毒同时对DC也有一定作用[8-9]. 国内尚未见相关报道. 我们以不同浓度LAM与CHB的DC共孵育, 观察LAM对CHB的DC生物学特性的影响, 为临床上LAM与DC疫苗联合治疗CHB寻找理论依据.

1 材料和方法

1.1 材料

rhGM-CSF, rhIL-4, 抗人CD1a、CD80、CD83、HLA-DR均购自美国R&D公司; RPMI 1640培养基, 美国GIBCO公司; 胎牛血清, 杭州四季青公司; MTT购自美国Sigma公司; 淋巴细胞分离液, 天津市津脉基因测绘技术有限公司; IL-12, IL-6 ELISA试剂盒, 上海森雄科技有限公司; LAM购自葛兰素史克(苏州)制药有限公司. 按中华医学会传染病与寄生虫学分会、肝病学分会联合修订的《病毒性肝炎防治方案》(2000-09西安)为诊断标准, 从门诊选出HBeAg(+)慢性乙型病毒性肝炎患者15例, 同时设健康对照者15例.

1.2 方法

常规方法分离CHB外周血单核细胞, 悬浮后加入24孔板, 37℃, 50 mL/L CO2孵箱温育2 h, 吸弃培养上清, 洗涤去除非贴壁细胞, 即获得贴壁细胞. 每孔加入含100 mL/L自体血清, GM-CSF 10 μg/L, IL-4 5 μg/L的RPMI 1640培养基1 mL, 于37℃, 50 mL/L CO₂孵箱培养[10], 并于第4天将DC分成2组: 一组加入LAM (0.125, 0.25, 0.5, 1, 2 mmol/L)作为实验组即LAM处理组; 一组继续培养作为对照组即LAM未处理组. 各组均隔天半量换液. 第8天收集细胞行相关检测.

1.2.1 DC形态学及表型检测: 倒置相差显微镜动态观察DC形态学变化并收集8 d的各组DC, 调细胞密度为1×10⁸/L, 各取细胞悬液0.5 mL加入FCM测定管, 分别加入FITC标记的鼠抗人CD1a和鼠抗人CD83, PE标记的鼠抗人CD80和鼠抗人HLA-DR各2 μL, 轻轻摇匀2 s, 室温避光反应45 min, 生理盐水洗涤1次, PBS重悬细胞后上机检测DC表型分子CD1a, CD83, CD80及HLA-DR的表达.

1.2.2 混合淋巴细胞反应[11]: 取健康者外周血, 分离出单核细胞, 37℃, 50 mL/L CO₂孵箱温育2 h后吸取未黏附细胞为淋巴细胞, 调密度为1×10⁸/L; 收集培养第8天的各组DC并用丝裂霉素C (50 mg/L)孵育30 min, 调密度为1×10⁷/L. 按DC与淋巴细胞比例为1∶10混合作为实验孔种植于96孔板, 200 μL/孔, DC组及T淋巴细胞组为对照孔, 设3复孔, 用MTT法在570 nm处测各孔A₅₇₀值, 并计算刺激指数(SI) ＝ 实验组A值/(反应细胞对照组A值+刺激细胞对照组A值). 另按ELISA试剂盒说明书操作, 测定培养第8天各组DC上清中IL-12和IL-6含量.

统计学处理 用SPSS 10.0统计软件进行处理, 全部数据均采用mean±SD表示, 以α = 0.05为检验性水准, P<0.05具有统计学意义.

2 结果

2.1 DC形态和表型

倒置显微镜下, 培养24 h后可见细胞呈集落生长, 2 d后细胞数目明显增多,体积开始逐渐增大, 胞体拉长; 第5天观察部分细胞有多个短突起, 有的胞体四周细胞膜扩展成多方向长突起, 呈树枝状, 面纱状, 第7, 8天时可见培养液中漂浮着颜色较深、云雾状的大细胞或细胞团. LAM处理小剂量组(图1B)与LAM未处理组(图1A)相比, 变形细胞较少, 细胞数目变化不大. LAM处理大剂量组(图1F)于加药第2天镜下观察可发现培养液中出现大量颗粒状物质, 细胞数量减少, 考虑为受药物毒性作用所致裂解的细胞碎片(图1). 在DC+LAM 0.5 mmol/L组, DC表型分子CD83, CD1a, HLA-DR的表达最高, CD80与LAM未处理组相比无明显差异, 但其膜分子表达量与健康人DC相比仍有所降低; 而其他组别与LAM未处理组相比, 虽有差异但无统计学意义. 说明CHB的DC体外与LAM (0.5 mmol/L)共孵育后, 其递呈抗原及刺激T细胞增殖能力相对增强(表1).

表1 LAM处理后树突状细胞表型分子的表达(mean±SD, ％).

	CD83	CD80	CD1a	HLA-DR
LAM 0 mmol/L (CHB)(A)	12.8±2.1	35.8±2.4	33.6±3.1	52.8±2.5
0.125 mmol/L	13.4±2.2	37.7±3.2	40.3±2.1	53.7±3.3
0.25 mmol/L	18.3±3.2	41.1±3.6	53.1±2.8	67.8±4.2
0.5 mmol/L (B)	20.2±2.5ae	41.7±4.2	54.1±4.2ae	74.5±5.2ae
1 mmol/L	12.8±2.3	25.7±3.1	16.6±1.9	42.1±3.9
2 mmol/L	11.9±1.3	24.7±2.6	16.5±1.8	41.0±3.5
0 mmol/L (健康人)(C)	61.4±6.6c	49.3±6.9	71.1±6.7c	87.3±6.5c

^aP<0.05,

^cP<0.05 vs A组;

^eP<0.05 vs C组.

图1 培养8 d树突状细胞形态(×400). A: LAM未处理组; B: DC+LAM 0.125 mmol/L; C: DC+LAM 0.25 mmol/L; D: DC+LAM 0.5 mmol/L; E: DC+LAM 1 mmol/L; F: DC+LAM 2 mmol/L.

2.2 淋巴细胞增殖能力

在DC+LAM 0, 0.125, 0.25, 0.5, 1, 2 mmol/L组DC刺激同种异体T淋巴细胞增殖能力分别为1.2±0.5, 1.3±0.6, 1.5±0.5, 1.9±0.6, 0.9±0.4, 0.6±0.3; DC+LAM 2 mmol/L组, 由于大量细胞裂解刺激淋巴细胞增殖能力减弱; 而DC+LAM 0.5 mmol/L组其刺激指数(SI)值与对照组相比有差异. 另经ELISA检测, 在LAM浓度依次为0, 0.125, 0.25, 0.5, 1, 2 mmol/L时, 与其共孵育的DC培养上清液中IL-12含量分别为268.1±34.3, 380.3±51.2, 500.4±89.4, 810.2±91.5, 255.1±73.1, 138.0±27.9 ng/L; IL-6含量依次为55.2±7.4, 47.1±5.2, 32.2±2.7, 28.4±2.6, 69.1±8.7, 67.3±8.4 ng/L; 健康人DC所分泌的IL-12, IL-6含量依次为1075.3±92.6, 21.2±6.7 ng/L(图2). 其中IL-12含量在DC+LAM 0.5 mmol/L组较高; IL-6含量在此浓度下较低, 与LAM未处理组及健康者DC分泌的IL-12, IL-6相比均有差异. DC+LAM 2 mmol/L组, 由于大量细胞裂解分泌IL-12, IL-6含量下降.

图2 LAM处理的DC分泌IL-12, IL-6水平(mean±SD).

3 讨论

HBV转基因小鼠试验中证明, 在清除乙肝病毒过程中, 机体免疫及细胞因子发挥了重要作用[12]. 感染后首先启动一系列非特异性早期应答, 包括干扰素, NK细胞及枯否氏细胞的活化; 彻底清除病毒则有赖于HBV特异性T细胞[13-14]. HBV分泌的各种抗原可刺激机体产生多克隆的、HBV抗原特异性的CTL应答, 杀伤携带HBV的靶细胞. 若不能对病毒进行有效的清除, 则造成感染的慢性化, 诱导机体内HBV抗原特异性的CD8+T细胞大幅度下降, 导致机体内抗原特异性T细胞免疫耐受, 造成CHB的发生[15-16]. 机体内的APC, 尤其是DC是特异性免疫应答的关键细胞, 启动和介导特异性T细胞免疫应答[17]. HBV感染慢性化与被感染者机体免疫功能低下尤其与其体内DC低表达黏附分子, 递呈抗原及刺激T细胞增殖能力低下有关[18-19]. 选择性修饰DC, 有效激活机体抗HBV特异性免疫反应, 已成为CHB治疗中的一条重要途径[20-22]. Beckebaum et al[9]报道, LAM在抗HBV同时, 能提高DC表面HLA-DR表达. 在此基础上, 我们参考LAM的临床应用剂量, 用不同浓度的LAM与培养第4天的DC共孵育, 第8天收获各组细胞进行了检测, 观察LAM对CHB的DC生物学活性的影响.

DC摄取抗原并将其处理成具有免疫原性的多肽, 以MHC-抗原肽复合物的形式表达于其表面的同时, 其膜表面的协同刺激分子与T细胞表面相应配体结合, 进而激活抗原特异性T细胞, 促其活化增殖, 产生免疫应答[23-25]. 实验中分别检测了DC表面CD1a, CD83, HLA-DR及CD80的表达. CD1a是DC的特异性表面标志, CD1a阳性细胞数的多少可反映DC的数量; CD83为DC的成熟性标志; HLA-DR为MHC-Ⅱ类分子之一, 主要参与抗原提呈; CD80为DC表面的共刺激分子之一, 通过与淋巴细胞表面的共刺激分子受体(如CD28)相结合, 形成强烈的共刺激信号, 参与T细胞的活化[26-27]. 本实验结果表明, LAM (0.5 mmol/L)与DC共孵育后提高了DC表面CD1a, CD83, HLA-DR的表达. 其CD80的表达与LAM未处理组相比有所增高, 但统计学上无差异. 与健康者DC相比, 其膜分子表达量仍有所降低. 而适当浓度LAM (0.5 mmol/L)孵育的DC淋巴细胞增殖能力增强, 这初步提示淋巴细胞的活化增殖不是单纯依赖于传统的双信号机制, 可能有其他机制参与. DC通过分泌IL-6, IL-12, IFN-γ等来控制Th0细胞向Th1和Th2细胞分化[28]. IL-12为成熟DC所分泌, 调节Th0细胞向Th1细胞分化, 促进Th1细胞分泌IFN-γ, IL-2等刺激T细胞活化增殖, 介导细胞免疫应答; IL-6主要由未成熟DC分泌, 主要促使Th0细胞向Th2细胞分化, 通过抑制Th1细胞的功能来抑制细胞免疫, 诱导免疫耐受[29-30]. 实验发现LAM可影响DC分泌IL-12, IL-6水平. 在DC+LAM 0.5 mmol/L组细胞培养上清液中IL-12分泌水平较高, IL-6分泌量较低. 故选择合适浓度LAM处理DC可促使T淋巴细胞增殖能力增强并向Th1分化, 增强其细胞免疫学活性. 实验结果表明, LAM可以提高CHB的DC表面分子CD1a, CD83, HLA-DR的表达, 提高DC分泌细胞因子IL-12水平, 降低IL-6分泌, 增强DC刺激淋巴细胞增殖能力. 表明LAM除了作为抗病毒药物外, 还可作为免疫调节剂, 一定程度上改善CHB的DC生物学活性. 这为临床上LAM与DC疫苗相结合进行慢性乙型肝炎的免疫治疗提供了理论依据.

评论

背景资料

DC是功能最强的抗原提呈细胞, 能激活初始化T细胞, 诱发T细胞免疫应答. CHB体内DC功能低下, 不能充分提呈抗原给T细胞, 使T细胞活化. 如何增强DC的抗原提呈功能是CHB治疗中需解决的关键性问题. LAM为一口服核苷类药物. 在细胞内经磷酸化后, 与dCTP竞争进入病毒合成中的DNA链, 终止病毒DNA的复制. 国外研究发现LAM在抗病毒同时能提高DC表面HLA-DR的表达, 国内尚未见相关文献报道.

创新盘点

本实验首次以不同浓度LAM与CHB的DC共孵育, 通过一系列指标检测来观察LAM对CHB的DC生物学特性的影响, 为临床上LAM与DC疫苗相结合治疗慢乙肝寻找理论依据.

同行评价

本文题目确切, 结果明确, 对于3TC可能存在的对于DC的免疫调节作用有一定的阐释作用.

备注

电编:张敏 编辑:潘伯荣

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