The expression of cylindromatosis in oxygen and glucose deprivation and reperfusion induced primary cortical neuron necroptosis
10.3760/cma.j.issn.1674-6554.2013.08.006
- VernacularTitle:去泛素化酶在皮质神经元缺糖缺氧再灌注损伤后坏死样凋亡中的表达
- Author:
Tao FENG
;
Hongmei DING
;
Deqin GENG
- Publication Type:Journal Article
- Keywords:
Cylindromatosis;
Necroptosis;
Oxygen and glucose deprivation;
Primary cortical neuron
- From:
Chinese Journal of Behavioral Medicine and Brain Science
2013;22(8):692-695
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the location of CYLD in the neurons and explore the expression of CYLD in OGD/reperfusion-induced neuronal necroptosis.Methods Primary cortical neurons were cultured for 6days and neuronal purity was observed by double staining immunofluorescence of β3-tubulin and DAPI.The location of CYLD was identified by double staining immunofluorescence of NeuN,DAPI and CYLD using primary cortical neurons cultured for 14 days.Then,primary cortical neurons were divided into 8 groups:Control,EBSS,DMSO,OGD/reperfusion(0 h,2 h,6 h,8 h,12 h).Neurons were pretreated with zVAD-fmk for 30 min,OGD for 2 h and the levels of CYLD were evaluated after reoxygenation at different time points.The peak value(8 h) was chosen as reoxygenation time point.Neurons were divided into two groups as Control and OGD.The levels of CYLD were determined in both cytoplasm and nucleus after OGD 2 h and reoxygenation 8 h.Results The double staining immunofluorescence showed that neuronal cultured purity was about 70％ and the CYLD strongly expressed in nucleus but weakly in cytoplasm.The levels of CYLD increased gradually with different reoxygenation time and arrived at peak value after reoxygenation for 8 h (P ＜ 0.05),which was in accordance with the change of LDH (P ＜0.05) (Control (1.00±0.00),EBSS (1.07 ±0.03),DMSO (1.09 ±0.03),0h (1.40±0.12),2 h (1.74±0.08),6 h (2.25 ± 0.12),8 h (2.97 ± 0.15),12 h (3.01 ± 0.08)).The level of cytoplasm CYLD increased significantly in the OGD group (reoxygenation for 8 h)than that in control group (P＜0.05).But the level of nucleus CYLD had no difference between OGD and control group (P ＞ 0.05),which was in accordance with the results of immunofluorescence.Conclusion The CYLD in neurons cytoplasm is involved in necroptosis induced by OGD/deprivation and downregulating of CYLD has a protective effect on the brain injury resulted from ischemia/ reperfusion.
