Gilpin D A, Hsieh C C, Kuninger D T, Herndon D N, Papaconstantinou J
Shriners Burns Institute, Galveston Unit, University of Texas Medical Branch, USA.
Surgery. 1996 Jun;119(6):674-83. doi: 10.1016/s0039-6060(96)80192-9.
Eukaryotic organisms possess natural defense mechanisms that protect against stress stimuli. One such mechanism is the activation of families of stress response genes (e.g., the acute phase response). Transcription of many of these genes is regulated by the leucine zipper or bZIP proteins (CCAAT binding/enhancer binding proteins [C/EBPs]). The aim of this study was to show that the C/EBP transcription factor genes respond to thermal injury.
Age- and weight-matched male Buffalo, Sprague-Dawley, and Fischer 344 12- to 16-week-old rats (275 to 325 gm) received a 40% total body surface area scald burn. Total RNA was isolated from livers at 0, 2, 5, 12, 24, and 48 hours. Northern blot hybridization was performed with 32P-labeled C/EBP alpha, C/EBP beta, and C/EBP delta cDNAs. Relative amounts of each mRNA were determined by densitometric analyses. For Western analyses liver nuclear and cytoplasmic protein extracts were prepared from burned and control rats. Nuclear protein extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto a PVDF membrane, and detected by using an enhanced chemiluminescence detection kit.
Expression of C/EBP genes is regulated in response to 40% total body surface area scald burn. A simultaneous decrease in C/EBP alpha and an increase in C/EBP beta and C/EBP delta mRNA levels occur in response to thermal injury. Western analyses detect changes in C/EBP alpha and C/EBP beta pool levels that suggest a differential regulation of these genes in response to thermal injury.
The responses of C/EBP alpha, C/EBP beta, and C/EBP delta are similar in Buffalo, Sprague-Dawley, and Fischer rats. The induced level, however, of C/EBP beta mRNA was highest in the Sprague-Dawley strain and lowest in the Buffalo strain and correlates well with the mortality of these strains. Because C/EBP beta is associated with the transactivation of stress response genes, this may explain the intensity of the response in the susceptible strains. This agrees with our hypothesis that the higher degree of sensitivity of the Sprague-Dawley rat to stress relative to the Buffalo strain may be due to inherently higher levels of factors such as C/EBP whose functions are associated with activation of stress response genes.
真核生物拥有抵御应激刺激的天然防御机制。其中一种机制是应激反应基因家族的激活（例如急性期反应）。这些基因中的许多基因的转录受亮氨酸拉链或bZIP蛋白（CCAAT结合/增强子结合蛋白 [C/EBP]）调控。本研究的目的是表明C/EBP转录因子基因对热损伤有反应。
年龄和体重匹配的12至16周龄雄性布法罗大鼠、斯普拉格-道利大鼠和费希尔344大鼠（275至325克）接受40%体表面积的烫伤。在0、2、5、12、24和48小时从肝脏中分离总RNA。用32P标记的C/EBPα、C/EBPβ和C/EBPδ cDNA进行Northern印迹杂交。通过光密度分析确定每种mRNA的相对量。对于蛋白质免疫印迹分析，从烧伤和对照大鼠制备肝细胞核和细胞质蛋白提取物。核蛋白提取物通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离，印迹到PVDF膜上，并使用增强化学发光检测试剂盒进行检测。
C/EBP基因的表达受40%体表面积烫伤的调控。热损伤导致C/EBPα同时减少，C/EBPβ和C/EBPδ mRNA水平增加。蛋白质免疫印迹分析检测到C/EBPα和C/EBPβ库水平的变化，表明这些基因在热损伤时存在差异调控。
布法罗大鼠、斯普拉格-道利大鼠和费希尔大鼠中C/EBPα、C/EBPβ和C/EBPδ的反应相似。然而，C/EBPβ mRNA的诱导水平在斯普拉格-道利品系中最高，在布法罗品系中最低，且与这些品系的死亡率密切相关。由于C/EBPβ与应激反应基因的反式激活相关，这可能解释了易感品系中反应的强度。这与我们的假设一致，即斯普拉格-道利大鼠相对于布法罗品系对应激的更高敏感性可能是由于诸如C/EBP等因子的固有较高水平，其功能与应激反应基因的激活相关。
