PSD-95小干扰RNA沉默效率评估与功能研究
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摘要
前言:突触后致密物蛋白质95(PSD-95)参与调节的谷氨酸受体相关信号转导是神经病理性疼痛中枢敏化机制的重要环节。
目的:评估小干扰RNA(siRNA)对PSD-95基因的沉默效率以及PSD-95基因沉默对谷氨酸诱导的神经细胞毒性与信号转导的影响。
方法:将化学合成的大鼠PSD-95基因特异的siRNA转染小鼠神经母细胞瘤/大鼠神经胶质细胞瘤杂交瘤细胞(NG108-15细胞),利用实时定量PCR评估转染试剂、转染方式、时间、细胞密度、siRNA剂量等对沉默效率的影响。利用谷氨酸刺激PSD-95基因沉默的NG108-15细胞,检测细胞生长活力与信号通路蛋白质表达与磷酸化的改变。
结果:序列特异性最高的siRNA在最佳的转染条件下导致PSD-95基因表达水平下降91.5%;PSD-95基因沉默既增强了神经细胞对谷氨酸毒性耐受能力,又可抑制Ca~(2+)/钙调蛋白依赖的蛋白质激酶Ⅱα异构型的磷酸化。
结论:PSD-95-siRNA可能成为有效控制神经病理性疼痛的基因治疗方法。
Introduction:Glutamate receptor-associated postsynaptic signal transduction,which is modulated by postsynaptic density protein 95(PSD-95),participates in the central sensitization of neuropathic pain.
Objective:The PSD-95 gene silencing efficacy by small interfering RNA(siRNA) and its succedent influence on glutamate-induced toxicity or signal transduction are to be illustrated.
Methods:Gene-specific siRNAs of rat PSD-95 were synthesized chemically for in vitro transfection into mouse neuroblastoma and rat glioma hybridoma(NG108-15) cells.The contributions of transfection agents,transfection methods,time course,cell density and siRNAs final concentration to the silencing efficacy were evaluated by real-time PCR. PSD-95 gene silenced NG108-15 cells were further stimulated by glutamate,with either the cell viability or the expression and phosphorylation of signal proteins to be assayed.
Results:The siRNA,which is exclusively identical to rat PSD-95,decreased PSD-95 mRNA level by 91.5%under appropriate conditions.PSD-95 gene silencing enhanced cellular tolerance against the glutamate toxicity,meanwhile the phosphorylation of Ca~(2+)/Camodulin dependent protein kinaseⅡαisoform was attenuated.
Conclusion:All results suggested the potential of PSD-95 gene specific siRNA in relieving neuropathic pain.
目的:评估小干扰RNA(siRNA)对PSD-95基因的沉默效率以及PSD-95基因沉默对谷氨酸诱导的神经细胞毒性与信号转导的影响。
方法:将化学合成的大鼠PSD-95基因特异的siRNA转染小鼠神经母细胞瘤/大鼠神经胶质细胞瘤杂交瘤细胞(NG108-15细胞),利用实时定量PCR评估转染试剂、转染方式、时间、细胞密度、siRNA剂量等对沉默效率的影响。利用谷氨酸刺激PSD-95基因沉默的NG108-15细胞,检测细胞生长活力与信号通路蛋白质表达与磷酸化的改变。
结果:序列特异性最高的siRNA在最佳的转染条件下导致PSD-95基因表达水平下降91.5%;PSD-95基因沉默既增强了神经细胞对谷氨酸毒性耐受能力,又可抑制Ca~(2+)/钙调蛋白依赖的蛋白质激酶Ⅱα异构型的磷酸化。
结论:PSD-95-siRNA可能成为有效控制神经病理性疼痛的基因治疗方法。
Introduction:Glutamate receptor-associated postsynaptic signal transduction,which is modulated by postsynaptic density protein 95(PSD-95),participates in the central sensitization of neuropathic pain.
Objective:The PSD-95 gene silencing efficacy by small interfering RNA(siRNA) and its succedent influence on glutamate-induced toxicity or signal transduction are to be illustrated.
Methods:Gene-specific siRNAs of rat PSD-95 were synthesized chemically for in vitro transfection into mouse neuroblastoma and rat glioma hybridoma(NG108-15) cells.The contributions of transfection agents,transfection methods,time course,cell density and siRNAs final concentration to the silencing efficacy were evaluated by real-time PCR. PSD-95 gene silenced NG108-15 cells were further stimulated by glutamate,with either the cell viability or the expression and phosphorylation of signal proteins to be assayed.
Results:The siRNA,which is exclusively identical to rat PSD-95,decreased PSD-95 mRNA level by 91.5%under appropriate conditions.PSD-95 gene silencing enhanced cellular tolerance against the glutamate toxicity,meanwhile the phosphorylation of Ca~(2+)/Camodulin dependent protein kinaseⅡαisoform was attenuated.
Conclusion:All results suggested the potential of PSD-95 gene specific siRNA in relieving neuropathic pain.
引文
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