摘要
目的研究抑制Homer-1b/c基因表达对神经元的保护作用。方法采用RNA干涉技术,通过逆转录聚合酶链式反应和Western blotting分析抑制Homer-1b/c表达效果。免疫组化检测代谢性谷氨酸受体1a(mGluR1a)表达变化。共聚焦显微镜观察神经元细胞内钙含量变化。通过乳酸脱氢酶(LDH)活性测定,研究抑制Homer-1b/c表达对神经元损伤的保护作用。结果转染神经元后36 h,Homer-1b/c表达明显被抑制,mGluR1a仅出现在胞浆部分。转染组较对照组细胞内钙荧光强度单位明显下降(23.4±2.8 vs 51.8±7.5),两组间差异有显著的统计学意义(P<0.05)。各组神经元损伤前培养液LDH活性差异无统计学意义(P>0.05),损伤后24 h,对照组和空载体组LDH活性分别为(158±23.0)U/L和(145±25.1)U/L,而转染组为(93±20.1)U/L,与对照组及空载体组比较,差异有统计学意义(P<0.05)。结论 Homer-1b/c对于mGluR1a从胞体向树突部位转运以及受体在突触后膜锚定都具有重要作用。Homer-1b/c参与了细胞内钙释放,降低Homer-1b/c表达对神经元具有保护作用。
Objective To study the neuroprotective effects of Homer-1b/c gene knock down on injured neurons.Methods RNA interference technology was performed to knock down Homer-1b/c gene.Reverse transcription-polymerase chain reaction（RT-PCR） and immunoblotting were used to identify Homer-1b/c,and immunohistochemistry was used to detect metabotropic glutamate receptor 1a（mGluR1a） expression after small interfering RNA（siRNA） transfection.Calcium concentration was measured with laser scanning confocal microscopy.Analysis of neuroprotective effects was performed by detection of the lactate dehydrogenase（LDH） level in the medium.Results Expression of Homer-1b/c was inhibited and mGluR1a appeared only in cytoplasm 36 h after siRNA transfection.Calcium fluorescence intensity unit was 51.8±7.5 in neurons of control group,while was decreased to 23.4±2.8 after siRNA transfection with statistical difference between two groups（P0.05）.There was no statistical difference in LDH concentrations among different groups.LDH concentrations were increased by（158±23.0）U/L in control and by（145±25.1）U/L in transfection without siRNA groups compared with（93±20.1）U/L in siRNA transfected group 24 h after traumatic neuronal injury（P0.05）.Conclusion Homer-1b/c plays an important role in mGluR1a transportation from cytoplasm to dendrites and anchoring to postsynaptic membrane.Homer-1b/c participates in cellular calcium releasing and reduces the expression of Homer-1b/c,which performs the neuroprotection on neurons.
出处
《中华神经外科疾病研究杂志》
CAS
2011年第4期321-325,共5页
Chinese Journal of Neurosurgical Disease Research
基金
国家自然科学基金资助项目(30930093)
陕西省自然科学基金资助项目(2005C245)
