余 蓉,邱少红,李传珩,张 勤,简维国 (石首市人民医院检验科, 湖北 石首 434400)
HBV前S基因芯片检测研究
余 蓉,邱少红,李传珩,张 勤,简维国 (石首市人民医院检验科, 湖北 石首 434400)
新发明的芯片法检测前S基因缺失突变发现一个乙肝携带者体内往往存在多种突变,传统分析方法耗时耗力。经研究发现,单个PCR反应产物足够对前S基因缺失分型。个体基因克隆后的产物用于前S芯片法杂交检测,比传统DNA测序法省时经济。前S芯片为0.7cm2大小的尼龙膜,对于没有测序仪器的临床机构来讲更方便经济。同时,芯片自动检测多种前S克隆,更适合于对慢性乙肝病毒携带者大范围的突变扫描。双盲法对比芯片法和传统测序法,二者对前S突变的检出率相近。
前S基因缺失突变;传统基因序列检测法;芯片法
慢性乙肝病毒感染是世界范围内原发性肝细胞癌发病的一个重要原因[1-6],血液或体液为其主要传播途径。在癌症发展之前乙肝病毒数十年的持续存在,多在40岁及以上病人发生乙肝相关原发性肝细胞癌[6-7]。慢性乙肝病毒携带者长期对病毒的控制可能对原发性肝癌的发生起到抑制作用。用于检测慢性乙肝病毒携带者体内病毒状况的乙型肝炎病毒标记物包括DNA滴度、乙肝病毒表面抗原、核心抗原、以及e抗原[7-8]。标记物联合检测揭示了体内病毒的复制情况以及宿主肝细胞内病毒颗粒数量。乙肝病毒活动性复制以及高病毒滴度与乙肝病毒导致的肝脏病情如炎症、纤维化、硬化和肝细胞癌的严重性相符[9]。
图1 灰色部分标示前S1和前S2突变部位,图上的箭头指示HBV基因的起始位点
19世纪90年代末两种类型的因前S基因缺失突变而形成的大蛋白被发现,并被认为与原发性肝细胞癌高度相关[10-11]。大蛋白主要表达在慢性乙肝病毒感染的后期,病毒基因组整合进入宿主染色体以后[12-15]。这种突变基因翻译而成的大蛋白首先在毛玻璃样肝细胞中被发现,组织学证据提示其多出现在慢性乙肝病毒感染者及原发性肝细胞癌患者体内[16]。前S突变形成的大蛋白与包括肝硬化和原发性肝癌在内的严重肝脏疾病密切相关,可能原因是其参与了肝细胞癌变的发生发展过程[17-25]。前S基因突变包括前S1和前S2两种不同的突变(见图1)。突变基因翻译而成的蛋白在内质网聚集,引发了剧烈的内质网压力[26],进而导致氧化毒性和DNA损伤[27]。同时存在非内质网途径,突变的前S2蛋白能够延长肝细胞寿命[28]。与原癌基因(c-jun)激活域结合蛋白1(JAB1)作用,诱导肿瘤抑制基因(p27kip1)降解,细胞周期进行,细胞增长[30]。因此认为在乙肝相关的肝细胞癌发生过程中前S突变很关键。
图2 前S基因芯片 7mm×10mm/w 包含42个核苷酸探针
图3 A显示野生型大蛋白基因;B显示前S1突变蛋白基因,探针6、7信号阴性;C显示前S2突变蛋白基因,信号12、13信号阴性
在发现前S突变以后,众多相关研究结果报道了其在慢性乙肝病毒携带者体内的广泛存在[17-25]。突变的前S形成的大蛋白,尤其是前S2蛋白与包括肝癌在内的乙肝相关疾病的严重性高度关联。前S基因缺失突变扫描对于慢性乙肝病毒携带者诊治尤其重要,同时与其他乙肝标志物联合检测以评估发生原发性肝癌的风险。
慢性乙肝病毒携带者体内前S基因突变的确认通常需要许多实验步骤,因为突变的前S基因来源于野生的前S基因,一个携带者体内可能存在许多前S自然突变,需要逐一克隆和测序,直接导致大样本检测的困难。新发明的检测方法,省略传统的DNA测序步骤,缩短程序使检测时间由7d减少至3d。
DNA序列分析结果表明急性乙肝感染患者的前S基因突变率相对较低(为7%),乙肝持续感染病程中前S基因缺失突变率逐渐上升至37%,在原发性肝癌患者中突变率升至60%,乙肝携带者基因组内的前S突变易化了肝癌的发展。
前S基因芯片包含42个基因探针(见图2),检测野生型大蛋白基因,前S1基因突变蛋白,前S2基因突变蛋白[27]。野生型能与芯片上所有探针杂交(见图3A),前S1突变显示信号6、7探针缺失(见图3B),前S2突变显示探针12、13缺失(见图3C)。
传统基因序列检测法检测前S1基因 21个克隆,前S1基因芯片分析检测19个;前S1缺失突变检出率传统基因序列检测法为70%,芯片法为65%。对比结果显示芯片法可以提供敏感性分析。
新发明的芯片法检测前S基因缺失突变发现一个乙肝携带者体内往往存在多种突变,传统分析方法耗时耗力。经研究发现,单个PCR反应产物足够对前S基因缺失分型。个体基因克隆后的产物用于前S芯片法杂交检测,比传统DNA测序法省时经济。前S芯片为0.7cm2大小的尼龙膜,对于没有测序仪器的临床机构来讲更方便经济。同时,芯片自动检测多种前S克隆,更适合于对慢性乙肝病毒携带者大范围的突变扫描。双盲法对比芯片法和传统测序法,二者对前S突变的检出率相近。
研究结果表明急性乙肝感染HBV病毒高滴度,前S缺失突变率低;慢性乙肝感染后期,HBV病毒滴度小,前S缺失突变率较急性期明显升高,至乙肝相关原发性肝癌阶段,突变率甚至高至60%。提示前S缺失发生在HBV长期持续感染阶段,最终成为乙肝病毒携带者。因此假设前S突变是肝脏癌变的重要因素,前S在内质网的聚集导致内质网压力增加以及氧化毒性,引起DNA损伤和突变[26-27]。有研究表明前S2突变引起细胞周期素A过表达,引发细胞周期开始,细胞增长[28-29]。结果表明,突变大蛋白尤其是前S2型参与了癌症的发生过程。因此对于原发性肝癌的高危人群慢性乙肝携带者,检测前S基因缺失突变很重要。
除外前S基因缺失,HBV一系列标志物例如HBV 滴度检测、HbeAg也与慢性乙肝携带者罹患肝癌的风险相关[30-31]。乙肝病毒复制引起肝脏损伤、炎症,释放肿瘤坏死因子,易化了纤维化的进程以及肝细胞的增殖[7-8]。HBV蛋白HBX能与许多宿主因子交互反应,是病毒原癌蛋白[32-33]。HBX与细胞启动子结合反应,作用于反式调控元件[34];同时调节蛋白酶体的功能,调控细胞降解以及病毒蛋白的合成[35]。因此假设前S突变加强了HBX的有害作用,引起细胞增殖和基因组的不稳定性,从而易化了肝细胞癌的发生。
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[编辑] 何 勇
10.3969/j.issn.1673-1409(R).2012.02.035
R349.6
A
1673-1409(2012)02-R073-04
2011-11-30
余蓉(1969-),女,湖北监利人,副主任检验技师,硕士,主要从事临床免疫学及质量控制工作。