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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。
Objective To investigate the role of serum reactive oxygen species (SRF) in the localization of cortical neurons and neuronal necrosis-like apoptosis after hypoxia-glucose-hypoxia (OGD) reperfusion injury. Methods Cortical neurons were cultured on day 6 and immunofluorescence staining was performed to observe the expression of neuron specific markers of 4,6-linked amidine-2-phenylindole (DAPI) and β3-tubulin. The purity of cultured neurons ; ② On the 14th day of culture, SRF localization was observed by DAB staining; ③ On the 6th day of culture, the lentivirus (HIV viral vector) was given to interfere with the expression of SRF; ④ On the 14th day after culture, OGD model 2 h, 8 h reperfusion), pretreatment with broad-spectrum anti-apoptosis inhibitor (zVAD-fmk) 20μmol.L-130min. The cells were divided into: normal control group (control group); OGD / zVAD-fmk / SRF lentivirus negative control group (OZ-group); ODG / zVAD-fmk / SRF lentivirus positive group (OZ + group). The changes of SRF protein and lactate dehydrogenase (LDH) levels were observed by colorimetry and Western blot. Results SRF was located in the nucleus in neurons. SRF protein level in OZ + group was down-regulated after SRF interference (P <0.05). LDH levels in neurons in different treatment groups were consistent with those in SRF protein level (P <0.05). Conclusion SRF is involved in apoptosis of neuron after ischemia / reperfusion injury in cortical neurons and locates in the nucleus.