慢性丙型肝炎患者干扰素治疗前后HCV HVR1准种的动态变化

摘要

目的: 探讨感染HCV1b型慢型丙型肝炎患者HCV HVR1区准种的复杂性与干扰素疗效的关系, 以便对干扰素治疗疗效进行预测.

方法: 应用IFN治疗的慢性丙型肝炎患者共20例, 于治疗前、治疗后3 mo、6 mo留取血清, -70℃冻存待测. 治疗方案为: 干扰能３mu, 3次/wk, 肌肉注射, 共24 wk. HCV HVR1准种的检测采用单链构象多态性聚合酶链反应法(SSCP法).

结果: IFN治疗前, 35%(7/20)的患者表现为SSCP低复杂性(SSCP条带数≤3), 65%(13/20)表现为高复杂性(SSCP条带数>3). 治疗3 mo后, 4例患者HCV RNA阴转, 均发生在低复杂性组, 其余大部分患者SSCP条带数减少1-2条带. 治疗6 mo时, 7例患者HCV RNA阴转, 仅2例发生在高复杂性组. 治疗结束时13例(65%)无应答患者中, 8例患者SSCP条带数均较治疗前减少, 2例无变化, 1例恢复到治疗前水平, 2例较治疗前升高.

结论: HCV HVR1区准种的复杂性可以对IFN治疗进行疗效预测, 治疗前准种数少者可预测对IFN治疗有较好的应答.

关键词: N/A

Dynamic changes of HVR1 quasispecies in chronic hepatitis C after IFN therapy

Lin Zhang, Gui-Zhen Zhao, Ying Li, Li-Lan Shi

Lin Zhang, Gui-Zhen Zhao, Ying Li, Li-Lan Shi, Department of Infectious Disease, The Second Clinical College, China MedicalUniversity, Shenyang 110003, Liaoning Province, China

Correspondence to: Dr. Lin Zhang, Department of Infectious Disease, The Second Clinical College, China Medical University, 36 Sanhao Street, Shenyang 110003, Liaoning Province, China.

Received: June 8, 2002
Revised: June 25, 2002
Accepted: July 5, 2002
Published online: February 15, 2003

Abstract

AIM: To evaluate the correlation between the complexity of HCV HVR1 quasi-species and their response to IFN therapy in patients with chronic hepatitis C.

METHODS: Twenty patients with chronic hepatitis C received IFN therapy (3 mu/d, three times/week for 24 wk). Serum quasispecies complexity of HVR1 was analyzed by polymerase chain reaction mediated singl-strand conformation polymorphism (SSCP) before and 3 mo, 6 mo post-therapy, respectively.

RESULTS: Of 20 patients, 7 had low level of complexity (SSCP bands ≤ 3), 13 had high level of complexity (SSCP bands > 3). The rate of HCV RNA negative in low level complexity group was higher than that in high level complexity group. Patients with low level of complexity prior to therapy had good responsive to IFN; Serum HCV RNA in some patients did not convert to negative after IFN therapy, however, the number of SSCP bands decreased gradually.

CONCLUSION: HCV HVR1 quasi-species complexity is a predictive factor in response to IFN therapy, a statistically significant correlation between high level of complexity of HCV HVR1 quasi-species and lacking of response to IFN therapy in patients with chronic hepatitis C was found.

Key Words: N/A

0 引言

HCV基因组具有高度的变异性, 可以逃避机体的免疫反应, 使HCV感染极易慢性化[1-4]. 尤其在E2/NS1区存在两个高变区(HVR1和HVR2), 其中HVR1被认为机体免疫选择部位[5-8], 可刺激机体产生免疫反应, 而机体产生的免疫压力又促使其产生更多的变异, 与HCV持续感染和慢性化密切相关, 并给临床治疗带来困难. HCV变异除产生不同的基因型和亚型外, 另一重要的特征是在宿主体内以准种的形式存在[9,10]. 准种即同-株病毒, 基因组序列极为相似的病毒群, 但一些位点仍存在差异. 由于HVR1的高变性, 常用其来确定准种的数量[30]. 近年的研究发现, 基因型HCV1b型患者 HVR1区准种的复杂性(准种数)与干扰素治疗疗效有密切关系, 慢性丙型肝炎患者HVR1准种的复杂性程度越高, 对IFN无应答的可能性越大[11-16]. Sandres et al[17,18]则报道HVR1准种的复杂性与IFN疗效无关, 不能用于疗效预测. 我们对沈阳地区20例HCV1b型慢性丙型肝炎患者进行了IFN治疗前后HVR1准种复杂性的动态观察, 以探讨HCV HVR1准种的复杂性与干扰素疗效的关系, 报道如下.

1 材料和方法

1.1 材料

慢性丙型肝炎患者20例均感染HCV1b型、HCV RNA阳性, 男15例, 女5例, 年龄23-45岁. 干扰素治疗方案: 干扰能3 mu, 3次/wk, 肌肉注射, 疗程6 mo. 疗效判定: 治疗结束时, ALT正常、HCV RNA阴转为有效. 否则为无效. 分别于治疗前、后3 mo、6 mo留取血清, -70℃冻存备用.

1.2 方法

HCV基因分型采用C区型特异性引物PCR法(中国公共卫生1997; 13: 199-200). HCV HVR1准种检测采用SSCP法. 引物序列根据中国人基因组序列设计(病毒学报1993; 9: 114-126), 由华美公司合成. 外引物1: 5'-CTACTCCGGATCCCACAAGC-3'(核苷酸位置1 009-1 028); 外引物2: 5'-GGGCTCGGAGTGAAGCAATA-3'(核苷酸位置1 519-1 538). 内因物1: 5'-ATTCCATGGTGGGGAACTGG-3'(核苷酸位置1 085-1 104); 内因物2: 5'-GCAATACACTGGACCACACA-3'(核苷酸位置1 504-1 523). HCV RNA提取采用异硫氰酸胍-酚-氯仿提取法. 血清50 mL, 分别加入异硫氰酸胍200 mL, 氯仿/异戊醇50 mL, 振荡混匀, 离心, 13 000 r/min, 10 min, 取水相100 mL, 加等量异丙醇, 混匀, 离心, 13 000 r/min, 10 min, 弃上清, 控干残留液体, 加750 mL/L乙醇200 mL洗涤, 离心, 13 000 r/min, 5 min, 弃上清, 真空抽干, 65℃ 20 min备用.

HVR1基因片断PCR扩增: 逆转录及第-轮PCR扩增. 10×buffer 3 mL, dNTP3 μL, MgCl₂ 1.56 μL, Taq 2U, AMV 3U, Rnasin 20U, 外引物1 20pmoL(1 mL), 外引物2 20pmoL(1 mL), 水19 mL, 总反应体积30 μL. 37℃ 30 min逆转录之后进入PCR扩增, 反应条件为94℃ 300 s预变性, 94℃45 s、50℃ 45 s、72℃ 45 s, 共35个循环, 终延伸300 s. 第二轮PCR扩增. 取第-轮扩增产物5 mL, 10×buffer 3 mL, dNTP3 mL, MgCl₂1.56 μL, Taq酶2U, 内引物1 20 pmoL(1 mL), 内引物2 20pmoL(1 mL), 水15 μL, 总反应体积30 μL. 反应条件与第-轮相同. 取第二轮扩增产物5 mL进行2%琼脂糖凝胶电泳, 片断大小在280 bp左右为阳性结果.

SSCP检测: 取PCR产物10 μL, 加变性上样液10 μL, 进行96℃ 10 min预变性, 取出后立即放入冰浴中, 之后进行80 g/L聚丙烯酰胺凝胶电泳(300 V, 5 h), 胶取出后用100 μL/L乙醇加5 g/L冰乙酸进行固定20 min, 洗涤后用2 g/L硝酸银染色20 min, 之后用15 g/L NaOH加4 g/L甲醛显色, 肉眼观察至显带为止, 用100 μL/L冰乙酸进行终止. 将结果存入电脑.

2 结果

2.1 IFN治疗前后HVR1准种的动态变化

HCV RNA阳性的HCV1b型慢性丙型肝炎患者20例中, 7例(35%)表现为SSCP低复杂性, 13例(65%)为高复杂性, IFN治疗3 mo后, 4例(20%)患者HCV RNA阴转, 均发生在低复杂性组. 其余大部分患者SSCP条带数减少1-2条带. 治疗6 mo时, 7例(35%)患者HCV RNA阴转, 其中仅2例发生在高复杂性组. 13例无应答患者中, 8例SSCP条带数均有所减少, 2例无变化, 1例恢复到治疗前水平, 2例较治疗前增加(表1).

表1 IFN治疗前后HVR1准种的动态变化.

No	治疗前		3 mo		6 mo
	SSCP条带数	HCVRNA	SSCP条带数	HCVRNA	SSCP条带数	HCVRNA
1	2	+	0	-	0	-
2	4	+	2	+	1	+
3	3	+	1	+	0	-
4	5	+	5	+	5	+
5	4	+	2	+	1	+
6	6	+	5	+	3	+
7	2	+	0	-	0	-
8	3	+	3	+	3	+
9	4	+	3	+	4	+
10	5	+	3	+	2	+
11	4	+	1	+	0	-
12	6	+	3	+	3	+
13	2	+	0	-	0	-
14	4	+	4	+	5	+
15	1	+	0	-	0	-
16	4	+	2	+	0	-
17	4	+	2	+	2	+
18	3	+	1	+	1	+
19	20	+	20	+	5	+
20	5	+	20	+	3	+

2.2 HVR1准种复杂性与HCV RNA阴转率和疗效的关系

治疗结束时, 7例患者HCV RNA阴转, 5例(71.4%)发生在低复杂性组, 2例(15.4%)在高复杂性组, 二者相比具有显著差异(P<0.05). 治疗结束时, 20例患者中7例(35%)表现为完全应答, 13例(65%)为无应答. 有效组与无效组HVR1准种数的比较(2.43±0.98 vs 4.46±0.88, P<0.01)差异非常显著.

3 讨论

由于HCV基因组的高度变异性[19-22], 使HCV感染后很难被机体的免疫反应所清除, 80%以上的HCV感染者将转变成慢性肝炎, 并逐步发展成肝硬化和肝癌[23-26]. 目前尚无有效的疫苗预防HCV感染, IFN仍是唯-可以清除病毒、改善患者生化和组织学指标的药物. 然而, 慢性HCV感染者对IFN的完全应答率仅10-30%, 且副作用大, 治疗费用昂贵. 因此, 选择合适的病例进行治疗, 并对疗效进行有效预测, 具有重要临床意义. 在影响IFN疗效的众多因素中, 1 b型HCV感染是导致IFN疗效低下的重要因素之一[27-29]. 我国大部分HCV感染者为此种基因型. 近年在研究1 b型HCV抵抗IFN的机制时发现, HVR1准种的复杂性与IFN疗效有关, 大多数研究表明, HCV HVR1准种复杂性越高, 对IFN无应答的可能性越大. 但Sandres et al则认为, HVR1准种的复杂性与IFN疗效无关, 不能进行疗效预测. 我们的研究结果发现, 治疗前SSCP条带≤3组患者HCV RNA阴转率为71.4%, SSCP条带>3组患者为15.4%, 二者相比具有显著差异. 且治疗有效组SSCP条带数明显少于无效组. 表明HVR1准种的复杂性确与IFN疗效有关, IFN治疗前HVR1准种数较少者可预测对IFN有较高的应答率.

一般认为, IFN治疗3 mo时, HCV RNA仍未阴转或ALT仍未降至正常, 则该患者对IFN无应答的可能性较大. 6 mo时仍为此种情况则为无效, 应停药. 在对该组患者的动态观察中发现, 3 mo时有效率为25%, 6 mo时为35%. 部分患者虽然治疗结束时HCV RNA未阴转, 但SSCP条带数较治疗前减少. 由于该组患者均未延长治疗时间, 未能观察到延长疗程是否可以提高IFN应答率. 因此建议今后对治疗结束时无应答者, 但SSCP条带数在治疗过程中逐渐减少者, 可适当延长疗程.

在HCV准种的检测方法中, 以SSCP法最简单易行, 易于临床开展. 在此技术中, 对目的基因进行PCR扩增后, 产物通过加热变性获得单链DNA, 并通过电泳进行分析. 分离的单链的泳动速度取决于其三维构象. 因此众多异质性基因组成的混合体可被分离成不同的条带, 而且条带数即可代表基因序列的复杂程度. 尤其核苷酸片断<300 bp时, 更适用于此种方法.

总之, 对HVR1准种复杂性是否可以预测IFN疗效方面, 尚有不同的观点存在, 今后有必要进一步进行大样本研究和进行氨基酸序列测定以证实.

备注

编辑: N/A

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