The role of integration of HBV DNA into liver tissues in childhood on the development of hepatocellular carcinoma

大量的流行病学研究表明，肝细胞癌(肝细胞癌)高发地区和乙肝病毒(乙肝)流行地区之间存在显著的对应关系。最近，除了血清学证据外，分子杂交技术也证实了HBVDNA在肝细胞癌中的整合。然而，人们对乙肝病毒携带者的整合情况知之甚少，特别是在儿童时期。在这里，我们重点研究了肝细胞癌、慢性肝炎和儿童无症状携带者的肝组织中HBVDNA的存在。纯化的DNA用Hind III酶切，琼脂糖凝胶电泳，Southern印迹杂交分析。患者年龄5~15岁，5例慢性肝炎患者中有4例肝组织内HBVDNA整合。扩增片段大小在5.0~20kb之间，提示存在多个整合的HBVDNA群体。2例肝细胞癌(11岁，男，女)组织中均有HBVDNA整合。免疫印迹杂交显示其中1例出现清晰条带，显示肿瘤细胞的单克隆性生长，其余标本均可检测到7.6~20.0kb的弥漫性涂片。在2例无症状携带者中，1例HBVDNA也出现整合，通过分子克隆的方法，证实了后者的多重整合模式。在慢性活动性肝炎中，有几例患者出现宿主DNA重排，包括整合的前病毒DNA。这种重排可能对c-myc等细胞癌基因的激活很重要。从这些结果来看，大多数(如果不是全部)乙肝病毒携带者在童年早期就完成了HBVDNA的整合。对于乙肝病毒携带者，尤其是慢性肝炎患者，可能需要使用分子生物学技术进行长期随访，以早期预测乙肝病毒相关性肝细胞癌。

Numerous epidemiological studies indicated a striking correspondence between areas where frequency of hepatocellular carcinoma (HCC) is high and where hepatitis B virus (HBV) is endemic. Recently, besides serological evidence, the integration of HBV DNA in HCC has been demonstrated using molecular hybridization technique. However, little is known about the integration in HBV carriers, particularly in childhood. Here, we focused on the presence of HBV DNA in liver tissues of HCC, chronic hepatitis and asymptomatic carriers in childhood.The samples were obtained by biopsy and used for DNA extraction. Purified DNA was digested with Hind III, subjected to agarose gel electrophoresis and analyzed by the Southern blot hybridization technique. The age of patients are between 5-15.In 4 of 5 patients of chronic hepatitis, HBV DNA was integrated in the livers. Diffuse hybridization from 5.0 to 20 kb in size was observed, indicating multiple populations of integrated HBV DNA. All of 2 HCC cases (11 yrs old, male and female), HBV DNA was integrated in tissues. However, blot hybridization showed a sharp band in one, showing monoclonal growth of tumor cells, while diffuse smear from 7.6 to 20.0 kb was detected in the other samples. In 2 asymptomatic carriers, HBV DNA integration was also shown in one.Using molecular cloning method, multiple integration pattern was confirmed in the latter case. In the cases of chronic active hepatitis, rearrangement of host-DNA including integrated proviral DNA were found in several patients. This rearrangement may be important for the accivation of cellular oncogene, such as c-myc.From these results, the integration of HBV DNA was completed in early childhood in most, if not all, HBV carriers. For the early prediction of HBV-related HCC, long term follow-up using molecular biological technique may be necessary in HBV carriers, particularly in chronic hepatitis patients.