小鼠巨噬细胞α-肿瘤坏死因子基因表达的调节

Regulation of Tumor Necrosis Factor-alpha mRNA Expression in Murine Peritoneal Macrophages

本实验用液体杂交法,以人工合成的小鼠α-肿瘤坏死因子(TNF-α)mRNA互补的寡聚核苷酸为探针,观察活化小鼠巨噬细胞(Mφ)TNF-α基因的条件和机理。结果TNF-αmRNA在受到微量细菌脂多糖(LPS)刺激1h内的Mφ即得到表达,2h左右达高峰,以后逐渐下降,LPS仅刺激10min就足以使Mφ在2h的TNF-α mRNA表达与连续刺激2h的表达水平相似;LPS剂量在1～1000ng/ml范围内,其刺激作用呈剂量依赖性。钙离子载体A23187及Mφ活化因子本身不能诱导TNF-α mRNA活化,但与LPS的诱导作用有很强的协同效应。维甲酸对TNF-αmRNA的表达有一定的抑制作用,而三氟拉嗪则使表达明显增强。蛋白质合成抑制剂环己亚胺不仅对LPS的刺激有明显的协同效应,且本身也是TNF-αmRNA的强诱导剂;RNA合成抑制剂放线菌素D则强烈抑制LPS对基因表达的刺激作用。以上结果提示:①LPS诱导TNF-α基因的活化不能单以其升高胞内游离Ca^(2+)浓度来解释;②蛋白激酶C在TNF-α基因活化中可能起负反馈作用;③TNF-α基因的活化可能受细胞内某种代谢迅速的蛋白性抑制因子的调控。

The expression of tumor necrosis factor-alpha (TNF-α) mRNA in murine inflammatory peritoneal macrophages (Mφ) was studied with a sensitive liquid hybridization method. Upon exposure to 10 - 1000 ng/ml of lipopolysac-charide(LPS) , Mφ were induced to express TNF-a mRNA in a dose-dependent manner. mRNA was detectable within 1 h after stimulation, peaked at about 2 hand then gradually declined. A 10 min treatment with LPS was enough to stimulate the maximal level of TNF-a mRNA, as determined in a 2 h period. Although calcium ionophore A23187 and macrophage activating factor(MAF) (both can activate Mφ to mediate tumoricidal activity) did not induce TNF-a mRNA expression by themselves, they did act synergistieally with LPS. Treatment of Mφ with retinoic acid strongly inhibited LPS-induced TNF-a mRNA expression, whereas triflu-operazine had an opposite effect. Cycloheximide not only synergized with LPS but also induced TNF-α mRNA expression by itself. In contrast, actinomycin D completely blocked LPS-induced TNF-a gene activation. These findings indicate that LPS-induced TNF-a mRNA expression is not solely due to an increase in intracellular free calcium ion and is independent of the protein kinase C pathway of signal transduction. In addition, TNF gene activity may be regulated by short-lived protein repressor(s).