李永纲, 刘明旭, 王福生, 金磊, 洪卫国, 中国人民解放军解放军第302医院传染病研究所生物工程室  北京市  100039
李永纲, 男, 1964-11-24生, 天津市人, 汉族. 1989年第四军医大学毕业, 2002年军事医学科学院硕士研究生毕业, 主治医师, 主要从事传染病的临床及研究工作.
北京市自然科学基金重点资助课题, No.7011005
项目负责人:王福生,100039,北京市,中国人民解放军解放军第302医院传染病研究所生物工程室.  fswang @public.bta.net.cn
电话: 010-66933332    传真: 01063831870
收稿日期: 2002-06-20    接受日期: 2002-07-05

Genetic polymorphisms in IL-10 and IL-12b allele promoter regions in Chinese patients of Han nationality with HBV infection

Yong-Gang Li, Ming-Xu Liu,Fu-Sheng Wang, Lei Jin, Wei-Guo Hong

Yong-Gang Li, Ming-Xu Liu,Fu-Sheng Wang, Lei Jin,Wei-Guo Hong,Division of Bioengineering, 302 Hospital of Chinese PLA , Beijing 100039 China 
Supported by the Natural Science Foundation of Beijing, No.7011005
Correspondence to: Dr. Fu-Sheng Wang, Division of Bioengineering , 302 Hospital of Chinese PLA,Beijing 100039,China. fswang @public.bta.net.cn
Received: 2002-06-20    Accepted: 2002-07-05

Abstract
AIM: To investigate the association of genetic susceptibility to hepatitis B virus infection and disease progression with the allelic polymorphisms of IL-10 and IL-12b promoter regions in Chinese Han population.

METHODS: Two groups of indigenous Chinese subjects  (314 subjects in total) were recruited in this study. Group 1 included 104 unrelated patients with chronic hepatitis B virus infection and 76 unrelated healthy donors. Group 2 contained 134 related subjects from seven HBV-infected pedigrees of Han ethnic origin. Total genomic DNA samples were purified from the 1.5 ml of peripheral blood of all participated individuals by using the QIAgen purification DNA kit. Genotyping of IL10-5'A and IL12-5'C alleles was performed by means of PCR-RFLP (restriction fragment length polymorphism) and further proved by direct DNA sequencing. All data were statistically analyzed by using SAS software.

RESULTS: IL10-5'A mutant frequency in unrelated healthy subjects was 41.9 % compared with 42.1 % in unrelated HBV-infected patients, while IL12-5'C mutant frequency was 64.6 % and 55.8 % among healthy individuals and HBV-infected patients, respectively. No significant difference was found among the unrelated healthy individuals and unrelated HBV-infected patients. In related individuals from the seven HBV-infected pedigrees, the mutant frequency of IL12-5'C allele was found to be identical to that in unrelated healthy and HBV-infected patients, but the mutant frequency of IL10-5'A allele (19.5 %) was significantly different from that (42.0 %) in unrelated group (P <0.01).

CONCLUSION: The polymorphisms of IL10-5'A and IL12-5'C allele promoter regions were not correlated with hepatitis B virus infection and disease progression among unrelated subjects, but there was a significantly lower mutant frequency of IL10-5'A allele among related subjects.

Li YG, Liu MX, Wang FS, Jin L, Hong WG. Genetic polymorphisms in IL-10 and IL-12b allele promoter regions in Chinese patients of Han nationality with HBV infection. Shijie Huaren Xiaohua Zazhi  2003;11(8):1139-1143

摘要
目的: 我国是一个乙型肝炎大国，乙型肝炎感染及其转归显然有人种、人群差异. 本研究对我国汉族人群中乙型肝炎病毒(hepatitis virus B，HBV)感染和发病与白介素-12 p40(interleukin-12 p40，IL-12b)、白介素-10(interleukin-10，IL-10)启动子区基因多态性之间的关系进行了探讨.

方法: 利用聚合酶链反应-限制性内切酶片段长度多态性分析 (restricted enzyme-fragment length polymorphisms， PCR-RFLP) 的方法对非相关人群的健康人群与HBV感染患者(轻、中度慢性肝炎、重度慢性肝炎)和HBV感染家系中的HBV携带者、既往HBV感染者、健康人群的IL-12b，IL-10启动子区-540C、-592A ( IL10-5’A，IL12-5’C)单碱基多态性(SNP)进行了分析，并且对两个突变位点进行了基因测序. 使用SAS统计软件进行统计分析.

结果: IL10-5’A、IL12-5’C在非相关人群中的分布符合Hardy-Weinberg平衡. 非相关人群IL10-5’A、IL12-5’C突变等位基因在HBV患者中的频率近似于健康人群(IL10-5’A，HBV患者42.08 %，健康人41.9%，P >0.05; IL12-5’C ，HBV患者55.8%，健康人64.6%，P > 0.05). IL10-5’A与IL12-5’C基因分布在慢性肝炎(轻中)和慢性肝炎(重)两组无显著性差异(P >0.05). 提示IL10-5’A，IL12-5’C单碱基多态性(SNP)对乙型肝炎病情轻中无影响; HBV感染家系中IL10-5’A，IL12-5’C单碱基多态性在携带者、健康人群以及既往感染者三组间均匀分布，IL12-5’C突变频率与非相关人群相似(非相关人群-540T 55.3 %，相关人群-540T 55.9 %); IL10-5’A突变频率与非相关人群有显著性差异(非相关人群-592C 42.0 %，相关人群-592C 19.5 %，P <0.01).

结论: IL10-5’A突变比IL12-5’C突变在HBV感染家系中可能起到更重要的作用，或影响相关人群对HBV的易感性.

李永纲, 刘明旭, 王福生, 金磊, 洪卫国. 汉族人IL-12b和IL-10启动子区基因多态性与HBV感染的相关性. 世界华人消化杂志  2003;11(8):1139-1143
http://www.wjgnet.com/1009-3079/11/1139.asp
 
0   引言
免疫应答有关的细胞因子基因的多态性可能对HBV感染的结局产生一定的影响^[1]. 当细胞因子IL-12水平较低时，预先应用rIL-12治疗可能有助于提高干扰素治疗的应答率^[2]. 乙型肝炎患者的血清IL-10 ，IL-12水平与病情的轻重密切相关^[3-6]. IL-10对于IL-12的调控是抑制分泌. 不同种族体内细胞因子水平是不同的，个体间细胞因子的分泌量不同受其基因启动子区基因多态性的影响^[7-9]. 我们探讨了IL-12 p40启动子区-540C单核苷酸突变(SNP)位点即IL-12 p40C→A(IL12-5’C) 和IL-10启动子区-592A SNP位点即IL-10-592A→C(IL10-5’A)与乙型肝炎发病及易感性的关系.

1   材料和方法
1.1 材料  根据2000年全国传染病与寄生虫病学术会讨论修订的“病毒性肝炎防治方案”试行标准，我们将患者组分为慢性肝炎(轻中度)、慢性肝炎(重度)组. 我院诊断明确的乙型肝炎住院患者104例，血清HBsAg检查均阳性，男84例 ，女20例 ，年龄24-73(46±12)岁. 慢性肝炎 (轻中度)75例 ，慢性肝炎 (重度 )29例. 随机挑选我院健康体检人员，乙型肝炎病毒血清标志阴性者76例. 采集了7个家系的外周血全血标本共134份，分别来自我国河南、河北、山东、山西. 男75份，女59份，都是汉族，年龄在1-83(平均为33±19)岁. 由于血缘关系的存在故称之为相关人群. 以上所有人均为汉族.
1.2 方法  基因组DNA提取采用QIAGEN公司的基因组DNA提取试剂盒，自外周血200 ml中分离DNA作为PCR模板，具体步骤见参考文献^[10]. 聚合酶链反应-限制性片段长度多态性(polymerase chain  reaction-restricted fragment length  polymorphism PCR-RFLP)IL12-5’C  PCR-RFLP检测引物 :扩增IL-12 p40启动子区域包含-540位点的上、下游引物分别为  5’-CAGACTTACCATGCTGAGAAA 3’对应于-502～-52 3nt，21 mer)及 5’AGTGGGAATACCTCAGCGTAA 3’(对应于-758～-779 nt，20 mer)，扩增产物长度286 bp; PCR反应扩增体系为 2 5 mL，含4×2.5 mmol/L dNTP 1.5 mL，12.5 mmol/L上、下游引物各1mL10×PCR缓冲液2.5 mL，Taq DNA聚合酶0.3 mL，基因组DNA1.0 mL. PCR条件为94 °C 5 min变性，然后再94 °C 30 s，55 °C 30 s，72 °C 30 s，30个循环，72 °C 10 min; RFLP酶切体系为10 mL，buffer4  2.5 mL，BSA 0.1 mL，HhaI限制性内切酶0.2 mL，双蒸水2.7 mL，PCR产物6 mL，37 °C水浴2 h后，酶切产物以20 g/L琼脂糖凝胶电泳，观察结果(表1). IL10-5’A  PCR-RFLP检测引物: 扩增IL-1O启动子区域包含-592位点的上、下游引物分别为 5’-TACTCTTACCCACTTCCCCC-3’(对应于-719～-699nt，20 mer)及5’-GGGGACCCAATTATTTCTCA 3′(对应于-402～-422 nt，20 mer)，扩增产物长度314 bp. PCR反应扩增体系为 2 5 mｌ，含4×2.5 mmol/L dNTP 1.5 mL ，MgCl₂ 1.0 mL，上、下游引物各1.0 mL，10×PCR缓冲液 2 .5 mL，Taq DNA聚合酶0.3 mL，基因组DNA1.0 mL. PCR条件为94 °C 5 min变性，然后再94 °C 30 s，55 °C 30 s，72 °C 30 s，33个循环，72 °C 10min； RFLP酶切体系10 mL ，buffer C 1.0Μl，BSA 0.1 mL  RsaI限制性内切酶0.2 mL，双蒸水1.7 mL，PCR产物7 mL， 37 °C水浴3 h后，酶切产物以20 g/L琼脂糖凝胶电泳，观察结果(表1).

表1  IL12-5’C和 IL10-5’A基因型与PCR-RFLP电泳结果

wt: 野生型; mt: 突变型.

        统计学处理  在本组中国汉族人中见IL10-5’A基因型 ，组间频率比较采用R碈表c²检验或Fisher确切检验，并检验等位基因在群体、性别上的分布是否符合Hardy Weinberg平衡定律. 数据处理均SAS软件处理.

2   结果
2.1 慢性乙型肝炎IL10-5’A，IL12-5’C的等位基因频率  我们发现在中国汉族(非相关的)健康人及HBV患者中IL-10启动子区-592A→C突变是普遍存在的，大约有46.6 %的人发生-592A→C的突变即IL10-5’A基因突变频率，部分个体发生纯合突变 (-592C/C，14.9 %). 经检验符合Hardy-Weinberg平衡定律 (P >0.05). 健康人群IL10-5’A基因突变频率、基因型频率与HBV患者的相似(P >0.05，表2). 慢性肝炎(轻中)、慢性肝炎(重)2组IL10-5’A基因突变频率无显著性差异(图1). 在非相关人群中，通过比较健康人及HBV患者中IL12-5’C等位基因分布特点，发现IL12-5’C→T突变是普遍存在的. 我国汉族人群53.3 %存在-540C→T的突变即IL12-5’C基因突变频率，其中突变型纯合子(-540 T/T)频率占25.7 %. 健康人及HBV患者的IL12-5’C基因型及基因突变频率(表2). 经检验符合Hardy-Weinberg平衡定律 (P >0.05). 慢性肝炎(轻中)、慢性肝炎(重)2组IL12-5’C基因突变频率比较，无显著性差异; 健康人群IL12-5’C突变频率与乙型肝炎患者的相似(P >0.05，图2).
         为了证实酶切结果，我们分别对IL10-5’A、IL12-5’C  PCR产物做DNA测序，结果证实IL10-5’A、IL12-5’C突变正确(图3). IL10-5’A测序略.
2.2 相关家系人群HBV感染者的IL10-5’A、IL12-5’C基因型  对7个家系的外周血全血标本共134份进行了研究. 采用R×C表双向无序c²检验，研究IL10-5’A、IL12-5’C基因型在HBV携带者(HbsAg阳性)、既往HBV感染者(HbsAg阴性、抗-HbcAb阳性)和健康人群之间的分布无显著型差异(P  >0.05，表3); 而相关人群与非相关人群IL10-5’A基因型相差显著; IL12-5’C基因型两组间无显著性差异(表4).

1: 野生型纯和子(C/C); 2, 3: 突变型杂合子(C/T); 4, 5: 突变型纯合子(T/T); M: 分子质量标准
图1(PDF) IL-12 PCR-RFLP产物的琼脂糖电泳照片.
2, 4: 野生型纯和子(A/A); 1, 3: 突变型杂合子(A/C); 5, 6: 突变型纯合子(C/C); M: 分子质量标准
图2(PDF) IL-10 PCR-RFLP产物的琼脂糖电泳照片.
A 纯合野生型; B 杂合型; C 纯合突变型. 箭头所指为SNP位点, 其对应位置的上图为T即正链的A; 下图为G即正链C.
图3(PDF) IL12-5’C PCR产物测序结果.

表2  慢性乙型肝炎IL10-5’A、IL10-5’C 基因型及突变频率

P >0.05.

表3  IL10-5’A、IL12-5’C基因型在HBV感染家系中的分布

表4  相关人群非相关人群基因型分布情况

^bP <0.01 vs 非血缘相关人群.

3   讨论
我国是一个肝炎大国，HBV感染和发病具有如下特点:(1)同一种病毒(HBV)感染，不同的个体所产生的结局不同^[11-17]; (2)同一种病毒，不同人群产生的易感性不同^[18-21];(3)同一种抗HBV药物，在不同个体产生的疗效反应不同^[22-27]; (4)同一种HBV疫苗，不同的个体产生预防效果有差别^[28-32]. 仔细分析HBV感染和发病的特点，我们发现宿主遗传因素重要作用，在一定意义上说机体的遗传基因可能是影响乙型肝炎的发生、发展、疗效和预后最主要的因素^[15-23].
      我们的结果发现，IL10-5’A，IL12-5’C在人群中的分布符合Hardy-Weinberg平衡. 非相关人群IL10-5’A，IL12-5’C突变等位基因在HBV患者中的频率近似于健康人群(IL10-5’A，HBV患者 42.1 %，健康人41.9  %，P  >0.05; IL12-5’C ，HBV患者 55.8 %，健康人54.6 %，P > 0.05). IL10-5’A与IL12-5’C基因分布在慢性肝炎(轻中)和慢性肝炎(重)两组无显著性差异(P >0.05)，IL10-5’A，IL12-5’C SNP可能单独对病毒性肝炎病情无影响; HBV感染家系中IL10-5’A，IL12-5’C SNP在携带者、健康人群以及既往感染者三组间均匀分布(P >0.05)，IL12-5’C突变频率相关人群(-540T 55.9 %)与非相关人群(-540T 55.3 %)相似(P >0.05)，相关人群IL10-5’A突变频率(-592C 19.5 %)与非相关人群(-592C 42.0 %)有显著性差异(P <0.01)，表示IL10-5’A突变比IL12-5’C突变在HBV感染家系中可能起到更重要的作用，影响相关人群对HBV的易感性.

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