病毒性肝炎 Open Access
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2004-03-15; 12(3): 622-625
在线出版日期: 2004-03-15. doi: 10.11569/wcjd.v12.i3.622
HLA-II类基因与慢性乙型肝炎重型化的相关性
蒋业贵, 王宇明
蒋业贵, 王宇明, 中国人民解放军第三军医大学西南医院全军感染病研究所 重庆市 400038
蒋业贵, 男, 1966-12-20生, 安徽巢湖市人, 汉族, 2002年获第三军医大学内科学博士学位, 主治医师, 讲师. 主要从事肝炎、肝硬化和消化系肿瘤的研究, 发表论文30篇.
通讯作者: 蒋业贵, 400038, 重庆市, 中国人民解放军第三军医大学西南医院全军感染病研究所. jiangyegui@yahoo.com.cn
电话: 023-68754141
收稿日期: 2003-03-08
修回日期: 2003-03-25
接受日期: 2003-04-05
在线出版日期: 2004-03-15

目的: 探讨HLA-DRB1、-DQA1和-DQB1等位基因多态性与慢性乙型肝炎重型化之间的关系.

方法: 采用聚合酶链反应/序列特异性引物(PCR/SSP)技术对52例慢性乙型肝炎和32例性慢性重症乙型肝炎的HLA-DRB1、-DQA1和-DQB1等位基因多态性进行了分析.

结果: HLA-DRB1*1 001在慢性重症乙型肝炎组的等位基因频率明显高于慢性乙型肝炎组(14.1% vs 1.9%, x2= 19.2 997, Pc =0.0281, RR=9.78). HLA-DQA1和HLA-DQB1等位基因频率在慢性重症乙型肝炎组和慢性乙型肝炎组间差异无显著性.

结论: HLA-DRB1*1 001与慢性乙型肝炎的重型化有关, 可能是一个对判断病情和预后有价值的实验指标.

关键词: N/A

引文著录: 蒋业贵, 王宇明. HLA-II类基因与慢性乙型肝炎重型化的相关性. 世界华人消化杂志 2004; 12(3): 622-625
Association between HLA classⅡgenes and severity of chronic hepatitis B
Ye-Gui Jiang, Yu-Ming Wang
Ye-Gui Jiang, Yu-Ming Wang, Institute of Infectious Diseases. Southwest Hospital, Third Military Medical University, Chongqing 400038, China
Correspondence to: Ye-Gui Jiang, Institute of Infectious Diseases. Southwest Hospital, Third Military Medical University, Chongqing 400038, China. jiangyegui@yahoo.com.cn
Received: March 8, 2003
Revised: March 25, 2003
Accepted: April 5, 2003
Published online: March 15, 2004

AIM: To investigate the association between the polymorphism of HLA -DRB1, -DQA1 and -DQB1 alleles and severity of chronic hepatitis B.

METHODS: HLA-DRB1, -DQA1 and -DQB1 alleles in 54 patients with chronic hepatitis B and 32 patients with chronic severe hepatitis B were analyzed by using polymerase chain reaction/sequence specific primer (PCR/SSP) technique.

RESULTS: The frequency of HLA-DRB1*1 001 allele in the chronic severe hepatitis B group was markedly higher than that in the chronic hepatitis B group (14.1% vs 1.9%, x2 = 19.2997, Pc = 0.0281, RR = 9.78). No significant different allele frequencies of HLA-DQA1 and HLA-DQB1 alleles were observed between the chronic severe hepatitis B group and the chronic hepatitis B group.

CONCLUSION: HLA-DRB1*1 001 is closely associated with the severity of chronic hepatitis B, and may be a valuble marker of evaluating the prognosis of patients with chronic hepatitis B.

Key Words: N/A


0 引言

不同个体对乙型肝炎病毒(HBV)易感性的差异与个体的免疫特性有关, 而后者主要取决于人类主要组织相容性复合体(MHC) . 人类白细胞抗原(HLA)是MHC的基因产物. HLA复合体作为调节机体免疫应答的重要基因群, 与抗HBV免疫反应有着密切的关系, 某些特殊的HLA基因型可能影响着HBV感染的慢性化和重型化. 我们利用聚合酶链反应/序列特异性引物(polymerase chain reaction/sequence specific primer, PCR/SSP)技术通过对慢性乙型肝炎和慢性重型乙型肝炎患者中HLA-Ⅱ类基因多态性的分析, 从基因水平研究免疫遗传因素在慢性乙型肝炎重型化发病机制中的作用.

1 材料和方法
1.1 材料

我科2000年住院慢性乙型肝炎52例, 男43例, 女9例, 平均年龄(33±9岁). 慢性重型乙型肝炎32例, 男27例, 女5例, 平均年龄(35±9岁). 符合2000年第十次全国传染病与寄生虫学术会议修订的诊断标准, 均为重庆地区无血缘关系的汉族人.

1.2 方法

基因组DNA提取 采用ReadyPCRTM全血基因组DNA纯化系统快速提取基因组DNA, 试剂盒购自华美生物工程公司. PCR/SSP技术测定HLA-DRB1, -DQA1和-DQB1等位基因多态性 按Olerup(Tissue Antigens, 1992; 39: 225; 1993; 43: 119)方法设计针对HLA-DRB1, HLA-DQA1和HLA-DQB1第2外显子区域多态性的SSP共39条, 组成40对特异性扩增引物对, 分别鉴定HLA-DRB1*1-10等位基因型别、所有已知有表达的10种HLA-DQA1和13种HLA-DQA1等位基因型别. SSP由上海生工生物工程技术公司合成. PCR扩增产物加样至含溴化乙锭的20 g/L琼脂糖凝胶中, 15 V/cm凝胶电泳20 min, 经紫外透射下泳道中见明显DNA条带为阳性, 确定其基因型别. PCR反应假阴性质控 每一PCR反应中, 都含有一对扩增基因和一个内对照基因(人生长激素基因)的引物, 后者引物序列为: 5'-primer: 5 '-GCC TTC CCA ACC ATT CCC TTA-3', 3'-primer: 5'-TCA CGG ATT TCT GTT GTG TTTC-3', 扩增产物大小为429 bp. 每次扩增后, 人生长激素基因作为PCR扩增的阳性内对照, 均应有阳性条带产物, 如这一基因受抑, 可能出现假阴性结果, 同时还有利于纯合子基因型检测的准确性. 假阳性质控 根据扩增DNA片段碱基数判断.

统计学处理 等位基因频率的计算采用直接计算法, 两组间等位基因分布的差异用四格表法进行x2检验, 对x2>3.84者按Fisher法计算确切P值, 并计算校正P值(Pc), 相对危险性(RR)按Woolf公式计算.

2 结果
2.1 HLA-DRB1等位基因检测

慢性重型乙型肝炎组的HLA-DRB1*1001等基因频率(14.1%)明显高于慢性乙型肝炎组(1.9%), 二者相比差异显著(x2=19.2997, Pc = 0.0 281, RR=9.78, 表1). 其他等位基因频率在慢性重型乙型肝炎组和慢性乙型肝炎组间差异无显著性.

表1 慢性乙型肝炎和慢性重型乙型肝炎患者的HLA-DRB1等位基因频率.
DRB1等位基因慢性乙型肝炎 (n =52)
慢性重型乙型肝炎 (n =32)
x2PcRR
阳性(n)等位基因频率(%)阳性(n)等位基因频率(%)
0101/010310.9611.560.1231> 0.051.65
03011817.31914.060.3 826> 0.050.74
0401/04111312.50914.060.1 001> 0.051.17
0701/070287.6946.250.1 346> 0.050.79
0801/080465.7734.690.0 969> 0.050.79
09011615.39914.060.0 663> 0.050.88
100121.92914.0619.2 9970.0 2819.78
1101/110410.9611.560.1 231> 0.051.65
1201/12021514.42812.500.1 474> 0.050.82
1301/130210.9600.000.6 228> 0.050.00
1303/130410.9600.000.6 228> 0.050.00
1401, 140465.7734.690.0 969> 0.050.79
1402, 140300.0000.000.0 000> 0.050.00
1501/15021110.5869.380.0 709> 0.050.86
1601/160221.9211.560.0 299> 0.050.81
Blank32.8911.560.3 054> 0.050.53
2.2 HLA-DQA1等位基因检测

慢性乙型肝炎组和慢性重型乙型肝炎组的HLA-DQA1等基因频率相比差异无显著性(表2).

表2 慢性乙型肝炎和慢性重型乙型肝炎患者的HLA-DQA1等位基因频率.
DQA1等位基因慢性乙型肝炎 (n =52)
慢性重型乙型肝炎 (n =32)
x2PcRR
阳性(n)等位基因频率(%)阳性(n)等位基因频率(%)
010198.6546.250.3 500> 0.050.68
01022221.151218.750.1 901> 0.050.8 2
010354.8134.690.0 013> 0.050.97
010410.9611.560.1 231> 0.051.65
020132.8811.560.3 054> 0.050.53
03011514.42812.500.1 474> 0.050.14
030200.0000.000.0 000> 0.050.00
040110.9611.560.1 231> 0.051.65
05012725.962031.250.8 992> 0.051.54
06011211.5469.380.2 203> 0.050.77
Blank98.65812.500.7 262> 0.051.59
2.3 HLA-DQB1等位基因检测

慢性乙型肝炎组和慢性重型乙型肝炎组的HLA-DQB1等基因频率相比差异无显著性(表3).

表3 慢性乙型肝炎和慢性重型乙型肝炎患者的HLA-DQB1等位基因频率.
DQA1等位基因慢性乙型肝炎 (n =52)
慢性重型乙型肝炎 (n =32)
x2PcRR
阳性(n)等位基因频率(%)阳性(n)等位基因频率(%)
0201109.62710.940.0 859> 0.051.18
03013735.582031.250.6 802> 0.050.68
030265.7734.690.0 969> 0.050.79
03031514.42914.060.0 051> 0.050.97
040154.8134.690.0 014> 0.050.97
040210.9611.560.1 231> 0.051.65
050132.8846.251.1 748> 0.052.33
050276.7334.690.3 154> 0.050.67
050321.9211.560.0 299> 0.050.81
060176.7334.690.3 154> 0.050.67
060243.8557.811.5 036> 0.052.22
060321.9211.560.0 299> 0.050.81
060421.9223.130.2 524> 0.051.67
Blank32.8923.130.0 082> 0.050.65
3 讨论

不同个体在HBV感染后临床表型复杂多样, 从一过性的抗原血症、自限性的急性肝炎、慢性病毒携带、进行性肝损害乃至肝硬化, 形成了复杂的疾病谱. 宿主在HBV感染后不同的临床发展过程, 除了与病毒本身的因素有关外, 更重要的是由于不同个体对HBV所发生的免疫反应的不同[1-9], 而后者主要取决于MHC. HLA是MHC的基因产物, 是首次发现的与疾病有明确关系的遗传系统, 为目前已知的最复杂的人类基因复合体, 各基因座位均有众多的等位基因, 这些基因位于人第6号染色体短臂上P21.3带处, 占人体整个基因组的1/30 00[10-27]. 在HLA 128个功能基因中39.8%基因和免疫系统有关, 特别是Ⅱ类区域中几乎所有基因均显示免疫相关功能. HLA与疾病相关性的具体机制尚不清楚. HLA基因最显著的特征是具有明显的多态性, 其多态性的差异决定个体免疫应答的不同. 因此, 推测HLA系统的特定等位基因是否在某些疾病的发生上起重要作用. HLA复合体作为调节机体免疫应答的重要基因群, 与抗HBV免疫反应有着密切的关系, 某些特殊的HLA基因型可能影响着HBV感染的慢性化和重型化[28-32].

严重的肝细胞损伤构成了重型肝炎发生的病理基础. 目前, 乙型肝炎肝细胞损伤机制的研究主要集中在HBV基因变异, 包括前C区和S区变异. HBV DNA前C区1 896位变异导致HBeAg前体蛋白的合成中断, 造成HBeAg减少或消失, 使抑制性T细胞(Ts)活性受到抑制, 造成Ts对HBc/HBeAg特异性辅助性T细胞调节作用降低, 引起刺激HBc/HBeAg特异性CTL产生能力异常增强和T细胞等免疫性别分泌细胞因子能力异常增强, 而导致肝细胞坏死加重. 但并非所有的慢性重型乙型肝炎患者的HBV DNA都发生变异, 且HBV DNA发生变异是在机体免疫压力下造成的. 不同个体的不同免疫特性可能是慢性乙型肝炎重型化的根本原因. 我们研究发现, 慢性重型乙型肝炎组的HLA-DRB1*1 001的等位基因频率明显高于慢性乙型肝炎组, 两者相比差异显著, 说明该等位基因与慢性乙型肝炎重型化有关, 可能是一个对判断病情和预后有价值的实验指标. 进一步说明特异性遗传背景可增加或降低人群患慢性乙型肝炎的危险性. 但这种HLA与慢性乙型肝炎连锁不平衡关联性受HLA基因的极其高度的多态性的影响, 因此在加大样本的基础上还需进行慢性乙型肝炎家系的HLA基因的调查研究, 从而为探讨和揭示慢性乙型肝炎重型化的发病机制提供更有价值的科学依据.

编辑: N/A

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