State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.
Hepatobiliary Pancreat Dis Int. 2009 Aug;8(4):383-8.
There are more than 100 million asymptomatic HBV carriers (ASCs) in China and they are at a high risk of developing liver disease, which creates a serious health problem. But more than 90% of normal adults clear virus from primary HBV infection, so the difference of gene expression between ASCs and normal adults determines the differential outcomes. To identify differentially expressed genes in ASCs compared to normal adults, we used suppression subtractive hybridization to compare gene expression.
cDNA subtracted libraries were constructed by suppression subtracted hybridization from peripheral blood monocytes of ASCs and normal adults, the subtracted clones were prescreened by dot blot hybridization, and the levels of genes of interest were confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR).
One hundred and two and 96 positive clones were acquired from the A-N and N-A libraries, respectively, and 14 and 11 clones were identified by dot blot hybridization in the A-N and N-A libraries. Two genes were confirmed as differentially expressed between a set of ASC and normal adult samples by real-time RT-PCR.
Differentially expressed genes in peripheral blood mononuclear cells between ASCs and normal adults were isolated by suppression subtractive hybridization, and included some new genes. Of the upregulated genes in ASCs, checkpoint suppressor 1 is associated with DNA damage-induced cell cycle arrest. Perforin 1 is associated with inflammation. The information about such alterations in gene expression could be useful for elucidating the mechanisms of ASC pathogenesis.
中国有超过 1 亿的无症状乙型肝炎病毒携带者（ASCs），他们患肝病的风险很高，这是一个严重的健康问题。但是，超过 90%的正常成年人可以从原发性乙型肝炎感染中清除病毒，因此 ASC 和正常成年人之间基因表达的差异决定了不同的结果。为了鉴定 ASC 与正常成年人相比差异表达的基因，我们使用抑制性消减杂交来比较基因表达。
通过抑制性消减杂交从 ASC 和正常成年人的外周血单核细胞中构建 cDNA 消减文库，通过斑点印迹杂交对消减克隆进行预筛选，通过实时逆转录聚合酶链反应（RT-PCR）确认感兴趣基因的水平。
从 A-N 和 N-A 文库中分别获得了 102 个和 96 个阳性克隆，在 A-N 和 N-A 文库中通过斑点印迹杂交分别鉴定出 14 个和 11 个克隆。通过实时 RT-PCR 从一组 ASC 和正常成人样本中确认了两个基因的差异表达。
通过抑制性消减杂交从 ASC 和正常成年人外周血单个核细胞中分离出差异表达的基因，其中包括一些新基因。在 ASC 中上调的基因中，检查点抑制剂 1 与 DNA 损伤诱导的细胞周期停滞有关。穿孔素 1 与炎症有关。这些基因表达改变的信息可能有助于阐明 ASC 发病机制的机制。
