IFN治疗慢性丙型病毒性肝炎患者外周血单个核细胞HCV的变化

摘要

目的: 应用荧光定量反转录多聚酶链反应技术(RT-PCR)及免疫组化技术研究慢性丙型病毒性肝炎患者外周血单个核细胞(PBMC)丙型肝炎病毒感染并评价干扰素对外周血单个核细胞中HCV的作用.

方法: 20例慢性丙型病毒性肝炎患者血浆、PBMC中的 HCV RNA含量应用荧光定量RT-PCR检测, 应用免疫组化技术检测HCV NS3在PBMC中的表达; 荧光定量RT-PCR法检测8例血浆及PBMC均阳性患者接受-2b干扰素治疗; 对照组6例血浆及PBMC荧光定量RT-PCR法检测均阳性, 未用干扰素治疗. 比较治疗前后用药组与对照组血浆及PBMC中HCV RNA载量变化. 应用双侧Wilcoxon秩和检验(wilcoxon 2-sample test), 确切概率法(fisher's exact test)进行数据分析.

结果: 20例中荧光定量RT-PCR检出血浆15例 、PBMC9例阳性；免疫组化法检出7例PBMC阳性. 应用确切概率法分析PBMC感染HCV RNA与血浆中病毒水平无相关性(P = 0.319). 8例患者接受干扰素治疗后其血浆及PBMC中HCV RNA载量均下降, 与治疗前相比有显著性差异(^aP = 0.0 017, ^bP = 0.0 059); 与对照组相比治疗后差异有显著性(^cP = 0.0 042, ^dP = 0.0 155).

结论: PBMC是丙型肝炎病毒肝外复制并表达的场所. PBMC感染HCV RNA病毒载量与血浆病毒水平无关. 干扰素对PBMC感染的HCV RNA有清除作用.

关键词: N/A

Alterations of HCV in peripheral blood mononuclear cells from IFN-treated patients with chronic HCV RNA infection

Li-Juan Fu, Shu-Lan Lü, Xian-Feng Cheng, Xiao-Yan Wang

Li-Juan Fu, Shu-Lan Lü, Xiao-Yan Wang, Department of Infectious Diseases, The Second Hospital of Harbin Medical University, Harbin 150076, Heilongjiang Province, China

Xian-Feng Cheng, Heilongjiang Provincial Hospital, Harbin 150001, Heilongjiang Province, China

Supported by: the Provincial Natural Science Foundation of Heilongjiang, No. GC02C159.

Correspondence to: Dr. Shu-Lan Lü, Department of Infectious Diseases, The Second Hospital of Harbin Medical University, 456 Xuefu Street, Harbin 150076, Heilongjiang Province, China. lijuan_fu_8@hotmail.com

Received: October 21, 2003
Revised: November 5, 2003
Accepted: December 16, 2003
Published online: March 15, 2004

Abstract

AIM: To study HCV RNA and HCV protein expression in peripheral blood mononuclear cells (PBMC) and to evaluate the therapeutic efficacy of alpha 2b interferon on the treatment of HCV RNA in PBMC of patients with chronic hepatitis C.

METHODS: Fluorescence quantitative RT-PCR was used to detect the quantitation of HCV RNA in PBMC and plasma, and immunohistochemistry assay was applied to identify HCV NS3 protein expression in PBMC from 20 patients with chronic hepatitis C; Eight patients with chronic hepatitis C received IFN therapy (5 MU/d, three times/week for 16 wks). HCV RNA load in plasma and PBMC was tested with a quantitative assay before and after treatment for 16 wks and compared with that of the control. The data were analyzed by Wilcoxon 2-sample test and Fisher's exact test.

RESULTS: HCV RNA in plasma was found in 15 of 20 (75%) chronic hepatitis C patients and in PBMC was found in 9 of 20 (45%) by fluorescence quantitative RT-PCR. HCV NS3 protein expression was found in 7 of 20 (35%) chronic hepatitis C patients by immunohistochemical assay. HCV RNA loads in PBMC was uncorrelated with those in plasma.In 8 patients, there were significant differences of the decreasing of HCV-RNA loads in plasma and PBMC between before and after interferon treatment for 16 wks (^aP = 0.0 017, ^bP = 0.0 059).The loads of HCV in plasma and PBMC in patients were significantly lower than that of controls after interferon treatment for 16 wks (^cP = 0.0 042, ^dP = 0.0 155).

CONCLUSION: HCV can infect, replicate and express in PBMC. HCV RNA loads in PBMC is uncorrelated with those in plasma. HCV RNA in PBMC can be cleaned out by alpha 2b interferon treatment.

Key Words: N/A

0 引言

丙型病毒性肝炎是威胁人类健康的全球性疾病[1-8], 50%-70%会导致慢性化, 最终发展致肝硬化[9-11], 并与原发性肝癌的发病关系密切[12-15]. 丙型病毒性肝炎慢性化机制尚不清楚, 众多学者认为丙型肝炎病毒感染外周血单个核细胞、甲状腺、胰腺、骨髓、脑脊液、脾脏等肝外组织可能与其慢性化机制相关, 其中以感染外周血单个核细胞与其慢性化机制关系最为密切[16-17]. 检测PBMC中HCV RNA正链及HCV RNA负链只表明HCV-RNA的存在, 不能说明病毒载量的变化. 因此我们应用荧光定量RT-PCR方法检测PBMC中HCV RNA, 并对干扰素治疗前后病毒载量的变化进行分析, 从而进一步探讨HCV-RNA感染PBMC的意义.

1 材料和方法

1.1 材料

慢性丙型病毒性肝炎患者20例, 男16例, 女4例, 年龄39.5±16.5岁. 诊断标准符合2000-09西安传染病与寄生虫病学术会议修订标准. 所有病例抗HCV(+), ALT(160±100 u/L), TBIL(12.3±6.7 mol/L), 并排除其他类型肝炎, 无饮酒及服用肝损害药物史. 其中8例荧光定量RT-PCR法血浆及PBMC均阳性患者接受干扰素治疗, 5MU隔日1次, 共16 wk; 对照组6例血浆及PBMC均阳性, 未用干扰素治疗. 淋巴细胞分离液(购于中国医学科学院血液研究所)、Trizol提取液(invitrogen公司)、HCV核酸扩增荧光检测试剂盒(购于达安公司)、NS3 mAb(购于北京病毒研究所)、SABC免疫组化试剂盒(购于天津灏洋生物有限公司)、DAB染色试剂盒(购于武汉博士德公司)、Roche LightCycler荧光检测仪、高速离心机

1.2 方法

EDTA抗凝静脉血4 mL, 离心取血浆0.5 mL用于提取血浆HCV-RNA, 剩余部分混匀应用淋巴细胞分离液梯度离心分离PBMC. 生理盐水洗涤2次, 按文献[18] 方法加胰酶及RNA酶以清除血浆HCV RNA污染. 洗涤定容2 mL, 1 mL用于滴片, 1 mL离心沉淀用于HCV RNA提取. Trizol提取液按说明操作提取PBMC总RNA, 于无水乙醇中-70 ℃冰箱保存. 两步法荧光定量试剂盒按说明操作, 荧光检测仪、软件定量分析. 琼脂糖凝胶电泳检测预期产物. 多聚赖氨酸预先处理玻片, PBMC混悬液涂片, 丙酮固定, 双氧水甲醇溶液浸泡, 1 g/L Triton X-100滴片, 抗NS3mAb滴片, 按SABC试剂盒、DAB试剂盒说明操作. 设置正常对照、空白对照及PBS替代对照.

统计学处理 应用双侧Wilcoxon秩和检验(Wilcoxon 2-sample test), 确切概率法(fisher's exact test)进行数据分析.

2 结果

慢性丙型病毒性肝炎患者20例中荧光定量RT-PCR检出血浆HCV定量(4.42×10⁵-1.05×10¹⁰copy/L)15例, 0copy/L 5例, 血浆阳性15例中PBMC检出8例(1.00×10⁴-2.20×10⁷copy/L), 血浆阴性5例中PBMC检出1例(1.02×10⁵ copy/L)并应用免疫组化法检测此例为阳性. 经统计分析PBMC中HCV RNA载量与血浆病毒水平无相关性(P = 0.319). 荧光定量RT-PCR检测血浆及PBMC均阳性8例患者接受干扰素治疗16 wk后其血浆及PBMCs中HCV RNA载量均下降, 与治疗前相比有显著性差异(^aP = 0.0 017, ^bP = 0.0 059), 与对照组相比治疗后血浆及PBMC中病毒载量差异有显著性(^cP = 0.0 042, ^dP = 0.0 155); 而血浆与PBMC中HCV-RNA载量评价干扰素疗效, 二者比较无显著性差.

应用免疫组化法检测PBMC中 HCVNS3 20例阳性7例. 阳性产物成棕黄色, 细颗粒状, 不均匀或呈弥漫分布定位于胞质和胞膜(图1, 2).

图1 外周血单个核细胞免疫组化. HCV NS3抗原阳性, 棕黄色物定位于胞质×400.

图2 外周血单个核细胞免疫组化. HCV NS3抗原阳性, 棕黄色物定位于胞膜×400.

3 讨论

丙型病毒性肝炎是一种广泛威胁人类健康的疾病, 陈良标et al[18-21]于PBMC中检测出完整病毒颗粒及HCV-RNA正负链, 表明PBMC是HCV-RNA肝外复制并表达的场所, HCV RNA亦可在PBMC中持续存在[22]. 但定性实验只能确定病毒的存在而不能对病毒复制的活跃程度进行分析, 因此我们应用荧光定量RT-PCR及免疫组化方法对20例慢性丙型病毒性肝炎患者进行检测. 其中血浆病毒载量检测出75%, PBMC检出45%, 免疫组化检出35%胞质及胞膜表达HCV NS3. 进一步证实丙型肝炎病毒可以感染慢性丙型肝炎患者外周血单个核细胞, 并在其中复制和表达. Meier et al[23] 认为是由于污染或被动吸附血浆中HCV RNA而导致PBMC检测HCV RNA阳性. 但是本实验经荧光定量RT-PCR检测血浆及PBMC中HCV RNA载量经Fisher's exact test, (P = 0.319), 表明PBMC中HCV RNA与血浆载量无关联. 本实验中1例患者血浆病毒载量为0copy/L, PBMC中检测出HCV RNA为(1.02×10⁵ copy/L)并且免疫组化检出此例PBMC中HCV NS3表达阳性, 表明HCVRNA可独立感染慢性丙型病毒性肝炎患者外周血PBMC. 同时我们发现检测PBMC感染HCV RNA荧光定量RT-PCR法与免疫组化法灵敏性相比无显著性差异, 但荧光定量RT-PCR法操作简便、节省时间更适用于临床应用.

干扰素治疗慢性丙型病毒性肝炎现已被公认为是较为有效的药物, 临床已应用于检测血浆病毒载量作为评价干扰素疗效的指标之一. 本实验采用荧光定量RT-PCR检测干扰素治疗(16 wk)前后PBMC中及血浆中HCV-RNA病毒载量变化. 结果干扰素治疗后血浆及PBMC中病毒含量均有下降, 与治疗前及对照组相比有显著性差异, 表明干扰素对于PBMC内的HCV-RNA也有清除作用[24-25]. 有研究表明慢性丙型肝炎患者PBMC感染HCV使CD4细胞比例减低, CD4/CD8比值降低甚至倒置[26], 从而影响机体细胞免疫机制, HCV RNA感染亦可引起机体细胞因子(IL, TNF等)失调, 使机体清除病毒能力降低, 导致病毒持续感染[27]. 而干扰素具有抗病毒和调节免疫双重功能, 探讨干扰素对慢性丙型病毒性肝炎患者外周血PBMC中HCV RNA清除的机制: (1)有研究表明干扰素可使CD4细胞增多从而增强机体的细胞免疫反应, 促使分泌IL-2、-IFN增加、IL-12降低, 从而增强了Th2细胞效应提高机体的抗病毒能力[28-30]. (2)干扰素可诱导PBMC双链RNA激活蛋白酶(PKR)mRNA及MxAmRNA表达生成抗病毒蛋白清除HCV RNA[31]. 本实验表明监测PBMC中HCV-RNA载量可作为评价干扰素治疗效果指标之一, 因有报道复发病例中HCV RNA再现于PBMC中比血浆中早, 故亦可作为干扰素治疗后复发的监测指标[32]. 但HCV RNA感染PBMC对于干扰素治疗效果的影响尚需结合病毒基因型、HCV-RNA水平、及HCV变异株等进一步研究[33-34].

总之, 应用荧光定量RT-PCR方法可证实HCV RNA可感染PBMC, 与免疫组化方法相比敏感性无差异, 但更适用于临床应用. 虽然应用荧光定量RT-PCR方法证实干扰素对PBMC中感染的HCV RNA有近期的清除作用, 但对远期的治疗效果尚需进一步随访观察.

备注

编辑: N/A

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