Study of Japanese flounder cytokine gene promoter functions

肿瘤坏死因子（TNF）是免疫调节和炎症反应的主要介质。然而，对鱼类中TNF介导的网络或信号级联知之甚少。本研究将日本拟鲽肿瘤坏死因子（TNF）基因的2.4 kb启动子区和绿色荧光蛋白（GFP）基因重组质粒分别导入日本拟鲽肾细胞系YO-K、日本拟鲽胚胎细胞系HINAE和斑马鱼受精卵中，研究TNF基因在细胞系和斑马鱼受精卵中的表达调控。将YO-K细胞和HINAE细胞与脂多糖（LPS）在20℃孵育24 h。在两种细胞系中，通过LPS刺激，表达GFP的细胞数量以及GFP蛋白的量增加。LPS刺激后，YO-K细胞和HINAE细胞中GFP mRNA的转录也被诱导。在YO-K细胞中，GFP的表达水平在刺激后3 h至6 h逐渐下降，而在HINAE细胞中观察到相反的趋势。当转基因斑马鱼胚胎暴露于LPS时，GFP在整个斑马鱼胚胎表面表达，Western blot分析也显示，在LPS浓度为140 μg/ml时，斑马鱼胚胎中表达的GFP蛋白水平高于LPS浓度为0和70 μg/ml时。这些结果表明，日本比目鱼TNF启动子调控的转录可能是在斑马鱼胚胎的LPS识别系统的控制下。

Tumor necrosis factor (TNF) is known as a primary mediator of immune regulation and inflammatory response. However, little is known about TNF-mediated networks or signal cascades in fish. In this study, we characterized the regulation of gene expression of Japanese flounder TNF in cell lines and zebrafish embryo.A recombinant plasmid containing the 2.4 kb promoter region of Japanese flounder tumor necrosis factor (TNF) and green fluorescence protein (GFP) was introduced into YO-K cells derived from Japanese flounder kidney, HINAE cells derived Japanese flounder embryos and zebrafish fertilized. YO-K cells and HINAE cells were incubated with lipopolysaccharide (LPS) at 20℃ for 24 h. The number of cells expressing GFP, as well as the amount of GFP protein was increased by LPS stimulation in both cell lines. GFP mRNA transcription was also induced by LPS stimulation in both YO-K cells and HINAE cells. In YO-K cells, expression level of GFP decreased gradually from 3 h to 6 h post stimulation, while a reverse trend was observed in HINAE cells. When the transgenic zebrafish embryos were exposed to LPS, GFP was expressed in the whole zebrafish embryonic surface and Western blot analysis also showed that the level of the expressed GFP protein in zebrafish embryo was higher at a LPS concentration of 140 μg/ml than at LPS concentrations of 0 and 70 μg/ml. These results indicate that transcription regulated by the Japanese flounder TNF promoter could be under the control of the LPS-recognition system in zebrafish embryos.