Baculovirus vectors repress phenobarbital-mediated gene induction and stimulate cytokine expression in primary cultures of rat hepatocytes
Baculovirus vectors repress phenobarbital-mediated gene induction and stimulate cytokine expression in primary cultures of rat hepatocytes
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DOI：
10.1038/sj.gt.3301246
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发表时间：
2000-08-01
期刊：
影响因子：
5.1
通讯作者：
Omiecinski, CJ
中科院分区：
文献类型：
--
作者：
Beck, NB;Sidhu, JS;Omiecinski, CJ
杆状病毒转染策略已被证明成功地将外源DNA转移到肝癌细胞和原代肝细胞中。当在培养的肝细胞中测试这些方法的实用性时，我们发现杆状病毒的存在破坏了苯巴比妥（PB）基因诱导过程，这是高度分化的肝细胞的一种强有力的转录激活事件特征，并抑制了白蛋白基因的表达。与我们实验室先前的报告一致，表明cAMP水平的增加可以完全抑制特异性细胞色素P450（CYP 1A 1）基因的诱导，在杆状病毒暴露后的原代肝细胞中测量cAMP浓度和PKA活性。然而，这两个参数都不受病毒存在的影响。为了评价杆状病毒暴露是否触发免疫应答调节，进行了RNA酶保护试验，并证明杆状病毒感染激活了原代肝细胞培养物中TNF-α、IL-1 α和IL-1 β的表达。免疫细胞化学实验表明，细胞因子的产生可能是由于存在少量的枯否细胞存在于培养群体。外源性添加的TNF-α也能有效抑制PB诱导，这与表明炎性细胞因子能够抑制生物转化酶系统表达的其他报告一致。比较研究证明了这些效应的特异性，因为肝细胞暴露于腺病毒载体不会导致肝基因反应性下调。这些结果表明，杆状病毒载体增强了原代肝细胞培养物中炎性细胞因子的表达，引起了人们的关注，这些特性是否会损害杆状病毒载体用于细胞色素P450基因调控研究以及人类肝脏定向基因治疗的使用。
Baculovirus transfection strategies have proven successful at transferring foreign DNA into hepatoma cells and primary hepatocytes. When testing the utility of these methodologies in cultured hepatocytes, we discovered that the presence of baculovirus disrupts the phenobarbital (PB) gene induction process, a potent transcriptional activation event characteristic of highly differentiated hepatocytes, and repressed expression of the albumin gene. In concert with previous reports from our laboratory demonstrating that increased cAMP levels can completely repress the induction of specific cytochrome P450 (CYP) genes, cAMP concentrations and PKA activities were measured in the primary hepatocytes subsequent to baculovirus exposure. However, neither parameter was affected by the presence of the virus. To evaluate whether immune response modulation was triggered by baculovirus exposure, RNase protection assays were performed and demonstrated that baculovirus infection activates TNF-alpha, IL-1 alpha: and IL-1 beta expression in the primary hepatocyte cultures. Immunocytochemical experiments indicated that the production of cytokines was likely due to the presence of small numbers of Kupffer cells present in the culture populations. Exogenously added TNF-alpha was also effective in repressing PB induction, consistent with other reports indicating that inflammatory cytokines are capable of suppressing expression of biotransformation enzyme systems. Comparative studies demonstrated the specificity of these effects since exposures of hepatocytes to adenoviral vectors did not result in down-regulation of hepatic gene responsiveness. These results indicate that baculovirus vectors enhance the expression of inflammatory cytokines in primary hepatocyte cultures, raising concerns as to whether these properties will compromise the use of baculovirus Vectors for study of cytochrome P450 gene regulation, as well as for liver-directed gene therapy in humans.