摘要
目的研究脑缺血后细胞外信号调节激酶(ERKs)对细胞周期调控的影响。方法建立光化学法诱导大鼠局灶性脑缺血模型,分为脑缺血组(对照组及治疗组)和假手术组。治疗组于缺血前30rain尾静脉注入U0126溶液,对照组尾静脉注入相同体积不含U0126的DMSO稀释溶液。应用免疫组织荧光化学法观察细胞周期蛋白D1(CyclinD11和细胞周期蛋白E(CyclinE)阳性细胞表达;免疫印迹(Westem blot)检测磷酸化ERK1/2(pERKl/2)、CyclinD1和CyclinE的蛋白表达;半定量逆转录-聚合酶链反应(RT—PCR)观察转录因子E2FmRNA的表达。结果治疗组CyclinD1和CyclinE阳性表达的细胞数较对照组显著减少fP〈0.051;缺血对照组pERK1/2蛋白表达显著强于治疗组(P〈0.05),4h时间点表达最为明显,12h时间点恢复到基线水平,CyclinD1和CyclinE表达6h开始升高,12h表达最为明显,治疗组较对照组显著减弱伊〈0.05);缺血对照组E2FmRNA的表达显著强于治疗组和假手术组伊〈0.05),以7d表达最为明显。结论EeKs在大鼠脑缺血中发挥重要作用,抑制脑缺血引起的ERK1/2磷酸化,可降低细胞周期蛋白CyclinD1、CyclinE和E2F的表达,即ERKs可影响细胞周期的调控。
Objective To investigate the effect of extracellular signal-regulated kinases (ERKs) on cell cycle regulation after ischemia. Methods Ischemic model was induced by photochemistry method. Animals were divided randomly into cerebral ischemia groups (control and treatment groups) and sham group. Rats in treatment group were subjected to U0126 solution injection at 30 min preischemia through caudal veins, and animals in control group were subjected to identical volume DMSO solution without U0126. Positive immunostaining for CyclinD1 and CyclinE were detected by immunohistofluorescence method. Expressions of phosphorylated ERK1/2 (pERK1/2), CyclinD1, and CyclinE proteins were examined by Western blot in ischemic slide of brain cortex. Semi-quantitative reverse transcfiption-polymerase chain reaction (RT-PCR) was used to detect the expression of transcription factor E2F mRNA in ischemic slide of brain cortex. Results The numbers of CyclinD1 and CyclinE positive cells were highly decreased in the U0126-treated group (P〈0.05 vs vehicle-treated group). Expression of pERK1/2 protein in the ischemic group was significantly higher than that in the U0126-treated group, which peaked at 4 h, and decreased to the baseline at 12 h after ischemia. While the expression levels of CyclinDl and CyclinE in the U0126-treated group were increased at 6 h post injury, peaked at 12 h after injury (P〈0.05 vs that in vehicle-treated group). In addition, expression of E2F mRNA in the vehicle-treated group was significantly higher than those in the sham-operated group and the U0126-treated group (P〈0.05). Conclusions ERK pathway plays a very important role in cerebral ischemia. Inhibiting ERK1/2 phosphorylation post-ischemia reduces the expressions of CyclinD1, CyclinE and E2F, which indicates that ERK can affect cell cycle regulation.
出处
《中华神经医学杂志》
CAS
CSCD
2008年第6期567-572,共6页
Chinese Journal of Neuromedicine
基金
国家自然科学基金(30230140)