慢性乙型肝炎与肝硬化患者乙型肝炎病毒S蛋白变异分析

doi:10.3969/j.issn.1673-8640.2020.05.007

摘要/Abstract

摘要：

目的探讨慢性乙型肝炎（CHB）患者、肝硬化患者乙型肝炎病毒（HBV）S蛋白变异的差异及其临床意义。方法选取CHB患者114例,其中慢性HBV携带者41例（慢性HBV携带组）、非活动性乙型肝炎表面抗原（HBsAg）携带者38例（非活动性HBsAg携带组）、肝硬化组35例（肝硬化组）。采用测序法检测所有对象的HBV S蛋白基因序列。结果慢性HBV携带组、非活动性HBsAg携带组与肝硬化组之间年龄和HBV DNA载量差异均有统计学意义（P<0.01）,性别及HBV基因型差异均无统计学意义（P>0.05）。肝硬化组HBV S蛋白总体变异率明显高于非活动性HBsAg携带组和慢性HBV携带组（P<0.05、P<0.01）。慢性HBV携带组、非活动性HBsAg携带组与肝硬化组S蛋白的主要亲水区（MHR）外变异率及细胞毒T淋巴细胞（CTL）+辅助性T淋巴细胞（Th）免疫表位区变异率依次升高（P<0.01）。3组之间S蛋白的MHR及位于MHR内的“a”决定簇、LOOP1和LOOP2的变异率差异均无统计学意义（P>0.05）,MHR外的非免疫表位区变异率差异亦无统计学意义（P>0.05）。慢性HBV携带组中有1例患者同时出现MHR外Th免疫表位区、CTL免疫表位区和非免疫表位区3个位点变异,1例患者出现MHR与MHR外Th免疫表位区2个位点变异,其他患者均为单点变异,且MHR外CTL+Th免疫表位区与非免疫表位区变异率相当,呈随机分布。非活动性HBsAg携带组有5例（13.15%）患者出现2个位点以上的变异,肝硬化组有9例（25.71%）患者出现2个位点以上的变异,且2个组的变异位点集中于MHR外CTL+Th免疫表位区。结论 HBV S蛋白MHR外,尤其是MHR外CTL+Th免疫表位区变异不仅与HBeAg阴性血清学状态相关,也与肝硬化相关。

关键词: 乙型肝炎病毒, S蛋白, 基因测序

Abstract:

Objective To investigate the difference of hepatitis B virus（HBV） S protein mutations in patients with chronic hepatitis B （CHB） and cirrhosis,and to assess its role in clinical application. Methods A total of 114 patients with CHB were enrolled,and they were classified into 3 groups,asymptomatic HBV carriers（41 cases）,inactive hepatitis B surface antigen（HBsAg） carriers（38 cases） and cirrhosis patients（35 cases）. DNA sequencing was used to determine HBV S protein gene mutation. Results There was statistical significance in age and HBV DNA loads of asymptomatic HBV carrier,inactive HBsAg carrier and cirrhosis groups（P<0.01）,and there was no statistical significance in sex and HBV genotypes（P>0.05）. The gene mutation frequency of HBV S protein was higher in cirrhosis group than those in asymptomatic HBV carrier group（P<0.05） and inactive HBsAg carrier group（P<0.01）. The mutation rates in outside major hydrophilic region（MHR） and cytotoxic T lymphocytes（CTL） +helper T cell（Th） epitopes were increased in the 3 groups（asymptomatic HBV carrier<inactive HBsAg carrier<cirrhosis patient）（P<0.01）. There was no statistical significance among the 3 groups in MHR and a determinant,LOOP1,LOOP2 located in MHR and non-immune epitopes located outside MHR（P>0.05）. Single amino acid substitution was found in the majority of asymptomatic HBV carriers except 2 patients. Of these,one had 3 amino acid substitutions in CTL+Th epitopes and non-immune epitopes located outside MHR,and another had 2 amino acid substitutions in MHR and CTL+Th epitopes outside MHR. The mutation rates in CTL+Th epitopes and non-immune epitopes outside MHR were comparable and random for asymptomatic HBV carriers,whereas more than 2 mutation sites were determined in 5 cases of inactive HBsAg carriers（13.15%） and 9 patients with cirrhosis（25.71%）. These mutation sites preferentially concentrated at the CTL+Th epitopes outside MHR. Conclusions High prevalence of outside MHR variants especially CTL+Th epitopes is associated not only with hepatitis B e antigen（HBeAg）-negative serostatus but also with severe liver diseases,particularly cirrhosis.

Key words: Hepatitis B virus, S protein, DNA sequencing

中图分类号:

R373.2

原永明, 章晓鹰, 顾超, 张珏, 孙学华. 慢性乙型肝炎与肝硬化患者乙型肝炎病毒S蛋白变异分析[J]. 检验医学, 2020, 35(5): 428-433.

YUAN Yongming, ZHANG Xiaoying, GU Chao, ZHANG Jue, SUN Xuehua. Analysis of hepatitis B virus S protein mutation in chronic hepatitis B and cirrhosis patients[J]. Laboratory Medicine, 2020, 35(5): 428-433.

图/表 4

参考文献 12

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引物名称	序列（5'~3'）	产物长度/bp
HBV DNA B型	F:CTTATCGAAGACTGGGGACCCTGTG	758
	R:CCATGAAGTTAAGGGAGTAGCC
HBV DNA C型	F:TTCTCGACGACTGGGGACCCTGCAT	756
	R:CCATGAAGTTAAGGGAGTAGCC

引物名称	序列（5'~3'）	产物长度/bp
HBV DNA B型	F:CTTATCGAAGACTGGGGACCCTGTG	758
	R:CCATGAAGTTAAGGGAGTAGCC
HBV DNA C型	F:TTCTCGACGACTGGGGACCCTGCAT	756
	R:CCATGAAGTTAAGGGAGTAGCC

组别	例数	年龄/岁	性别/例		HBV基因型/例		HBV DNA/（IU/mL）
组别	例数	年龄/岁	男	女	B型	C型	HBV DNA/（IU/mL）
慢性HBV携带组	41	27.00（25.00~30.00）	23	18	14	27	8.11（8.00~8.25）
非活动性HBsAg携带组	38	35.00（30.00~46.50）^*	21	17	23	15	3.21（3.15~3.26）^*
肝硬化组	35	52.00（45.00~60.00）^*#	26	9	17	18	4.88（3.73~6.00）^*#
统计值		69.963	3.542		5.534		87.556
P值		0.000	0.170		0.063		0.000

组别	例数	年龄/岁	性别/例		HBV基因型/例		HBV DNA/（IU/mL）
组别	例数	年龄/岁	男	女	B型	C型	HBV DNA/（IU/mL）
慢性HBV携带组	41	27.00（25.00~30.00）	23	18	14	27	8.11（8.00~8.25）
非活动性HBsAg携带组	38	35.00（30.00~46.50）^*	21	17	23	15	3.21（3.15~3.26）^*
肝硬化组	35	52.00（45.00~60.00）^*#	26	9	17	18	4.88（3.73~6.00）^*#
统计值		69.963	3.542		5.534		87.556
P值		0.000	0.170		0.063		0.000

组别	例数	S蛋白	MHR	“a”决定簇	LOOP1	LOOP2	MHR外	CTL+Th免疫表位区	非免疫表位区
慢性HBV携带组	41	10 （24.39）	6 （14.63）	6 （14.63）	6 （14.63）	0 （0）	5 （12.20）	3 （7.32）	3 （7.32）
非活动性HBsAg携带组	38	16 （42.11）	6 （15.79）	5 （13.16）	3 （7.89）	1 （2.63）	12（31.58）^*	9 （23.68）^*	6 （15.79）
肝硬化组	35	23 （65.71）^**#	4 （11.43）	3 （8.57）	3 （8.57）	0 （0）	20（57.14）^**#	18（51.43）^**#	6 （17.14）
χ²值		13.175	0.306	0.685	1.156	2.018	17.421	19.151	1.941
P值		0.001	0.858	0.710	0.561	0.365	0.000	0.000	0.379