Study of mechanism of action of cytokines and signaling molecules using adenoviral transient expression vectors

用腺病毒载体(ADV)系统研究了基因表达的效率、几种ADV介导的细胞因子信号的体内外效应以及基因治疗的模型。LacZ-ADV在几种细胞系和原代细胞中瞬时、剂量依赖地表达，启动子活性的强弱顺序为CAG&GT；EF-1a&GT；SRA。LacZ的表达水平与细胞腺病毒受体之一的细胞表面Vitronectin受体水平有很好的相关性。多种细胞因子表达的ADV(GM-CSF、IL-2、IL-4等)的感染导致细胞因子水平在体外培养(COS，HeLa)和体内注射的小鼠中出现瞬时峰值。ADV感染淋巴细胞的试验没有成功，因为淋巴细胞表达很低水平的玻璃体连接蛋白受体，感染效率非常低。MOI&GT；1000点的感染只导致几个高细胞毒性的细胞感染。GM-CSF-ADV感染原代骨髓细胞可形成非因子依赖的集落。我们还将未插入的ADV或添加GM-CSF、IL-4等基因的ADV注射到小鼠体内。注射滴度极高的病毒导致几天内死亡。GM-CSF载体感染后，小鼠脾、骨髓造血功能增强。我们还制备了GM-CSF受体a-链和b-链及其突变体、c-kit、c-fms、Jak激酶1、2、3及其显性-阴性突变体等信号分子表达载体，并正在研究它们对细胞生长和表型的影响。基于这三年的结果，我们现在可以对ADV的有效性和感染模式有一个大致的了解，并继续将信号分子转移到小鼠疾病模型的研究中。

Using adenoviral vetor (AdV) system, we have investigated the efficiency of gene expression, the effects of several AdV-mediated cytokine signals in vitro and in vivo, and the models of gene therapy.Infection of LacZ-Adv resulted in transient, dose-dependent expression in several cell lines and primary cells, showing that the order of strength of promoter activities was CAG>EF-1a>SRa. The expression levels of LacZ correlated well with the cell surface vitronectin receptor levels that are one of the cellular adenoviral receptors. The infection of several cytokine-expressing AdVs (GM-CSF,IL-2, IL-4, etc.) resulted in transient peaks of cytokine levels in vitro cultures (COS,HeLa) and in vivo injected mice. Trial of infection of AdV into lymphcytes was not successful, because lymphocytes express very low levels of vitronectin receptors and infection efficiency was very low. Infection at MOI>1000 resulted infection into only several % of cells with high cellular toxicity. Infection of GM-CSF-AdV into primary bone marrow cells resulted in factor-independent colony formation. We also injected AdVs without insert, or with GM-CSF,IL-4 cDNA,etc. into mice. Injection of very high titers of viruses resulted in death within a few days. Infection of GM-CSF vector resulted in enhanced hematopoiesis in spleen and bone marrow. We have also prepared several signaling molecule-expressing vectors, such as GM-CSF receptor a-and b-chains, their mutants, c-kit, c-fms, Jak kinase 1,2,3 and their dominant-negative mutants, and are now examining the effects of their expression on cellular growth and phenotypes.Based on the results of these three years, we can now obtain gross understanding of the efficiency and infection patterns of AdVs, and are continuing the studies of transferig signaling molecules into mouse disease models.