血清反应因子参与皮质神经元缺糖缺氧诱导坏死样凋亡的实验研究

The Role of Serum Response Factor in Oxygen and Glucose Deprivation Induced Primary Cortical Neuronal Necroptosis

目的探讨血清反应因子(SRF)在皮质神经元中的定位及缺糖缺氧(OGD)再灌注损伤后神经元坏死样凋亡中的作用。方法皮质神经元:①培养第6天,免疫荧光观察4,6-联脒-2-苯基吲哚(DAPI)核染及β3-tubulin神经元特异性标志物表达,验证培养神经元的纯度;②培养第14天,二氨基联苯胺(DAB)显色法观察SRF定位;③培养第6天,给予慢病毒(HIV病毒载体)干扰SRF表达;④培养第14天,制作OGD模型(OGD 2 h,复灌8 h),预给予广谱抗凋亡抑制剂(zVAD-fmk)20μmol.L-130 min。将细胞分为:正常对照组(对照组);OGD/zVAD-fmk/SRF慢病毒阴性对照组(OZ-组);ODG/zVAD-fmk/SRF慢病毒阳性组(OZ+组)。经比色法、Western blot等方法观察SRF蛋白和乳酸脱氢酶(LDH)水平的变化。结果 SRF在神经元中定位于细胞核;经SRF干扰后OZ+组SRF蛋白水平下调(P<0.05);不同处理组神经元分泌LDH水平与SRF蛋白水平变化一致(P<0.05)。结论 SRF参与皮质神经元缺血/再灌注损伤后神经元坏死样凋亡,定位于胞核。

Aim To investigate the location and the role of serum response factor （SRF） in neuron on oxygen and glucose deprivation （OGD）-induced necroptosis. Methods Primary cortical neurons were cultured for 6 days, neuron-specific marker β3-tubulin and DAPI staining nuclear observed neuronal purity. The location of SRF was observed by DAB method with cortical neurons cultured for 14 days. Neurons with SRF knock- down after SRF lentivirus infection at day 6 followed by pretreatment with zVAD-fmk （20 pmol.L-1） for 30 minutes at day 14, exposed to OGD for 2 hours, and reoxygenation （R） for 8 hours, and then detected the location of SRF and level changes of SRF protein and LDH in neurons through the Western blot, colorimetricmethod. Results The immun of luorescence of SRF stronly expressed in the nucleus of neurons. SRF was downregulated by lentivirus-SRF（P〈0.05）. The levels of LDH and SRF had the same changing patternin neurons（P〈0.05）. Conclusion SRF locates in nucleus and involves in oxygen and glucose deprivation and reperfusion induced primary cortical neuronal necroptosis.