致痫灶靶向TD-PF胶束介导KCNJ4调控Kir2.3/Cavelion1共轭分子内向整流效应的研究

Refractory epilepsy pathogenesis is quite complex, the relevant ion channels mechanism is now becoming a hot spot. This study attempts to apply earlier building TD- PF active targeting polymer micelle as the carrier, carry the reducing expressed KCNJ4 gene in intractable epileptogenic zone then transfect it to model rats. By in vitro and in vivo experiments, we want to observe target gene expression and behavior of rats, brain electrical physiological and cell excitability as well as the potassium channel conductance changes, before and after KCNJ4 gene transfection. Combineding the technology of siRNA interference which stimulated the KCNJ4 genetic degradation as contrast experiments, we are expected to reveal that the abnormal expression and function changes of inward rectifier potassium channel gene KCNJ4 and its encoded Kir2.3 subunits may be the bases on epilepsy discharge cycle; we also validate the downstream Cavelion1 molecules within assisting Kir2.3 rectification, driving potassium ion of the conjugate adjustment. If we can confirm the correlation between Kir2.3 with intractable epilepsy and the synergistic effect between Kir2.3/ Cavelion 1 conjugate molecules, it may provide us a complete understanding of the inward rectifier mechanism of refractory epilepsy, then further offer experimental foundation and theoretical basis to conduct the KCNJ4 gene and protein Kir2.3 as the new ideal of antiepileptic drugs sites.

难治性癫痫发病机制复杂，与其相关的离子通道异常正成为热点。本研究尝试应用构建合成的TD-PF主动靶向聚合物胶束作为载体，将前期发现表达下调的KCNJ4基因靶向转染至模型大鼠脑内致痫灶，通过体内外实验观察干预前后目标基因表达及大鼠行为学、脑电生理与细胞兴奋性及钾通道电导变化，结合siRNA干扰技术激发KCNJ4降解进行对比实验，可望揭示内向整流钾通道KCNJ4基因及其编码的Kir2.3亚单位表达和功能异常可能是痫样放电循环产生的关键分子与生理学基础；同时验证通路下游Cavelion1分子在协助Kir2.3发挥内向整流效应、驱动钾离子内移时的共轭调节作用。如能明确Kir2.3与难治性癫痫发生间的相关性以及Kir2.3/ Cavelion1共轭分子间的协同效应，则可能为完整理解难治性癫痫的内向整流相关机制、进一步将KCNJ4及Kir2.3作为抗癫痫药物的新型理想作用位点，提供实验基础与理论依据。

难治性癫痫的发病机制复杂，治疗效果欠佳；与其相关的离子通道异常正成为热点。本研究尝试对合成的TD-PF胶束进行改良，将前期发现表达下调的KCNJ4基因靶向至致痫灶，同时结合其他方法、如选择特异性针对Kir2.3的过表达质粒与抑制性质粒转染海马神经元，以及能够特异性开放Kir2.3通道的开放剂Tenidap通过体内外实验观察干预前后目标基因、蛋白表达变化及小鼠行为学、脑电生理，细胞兴奋性与通道电流变化；同时验证下游共轭分子Caveolin-1表达变化趋势。.实验结果如下：第一部分研究完成对原始TD-PF载药胶束的验证、同时改良该胶束用于包载基因片段的材料学特性，构建适合包载KCNJ4mRNA的PAMAM-PEG-Angiopep2呈递体系，成功摸索PAMAM-PEG-Angiopep2/KCNJ4复合物转染原代海马神经元的最优条件；检测上述复合物转染原代海马神经元后Kir2.3的表达；同时观察其作为KCNJ4传输载体在颞叶癫痫慢性期小鼠体内的生物分布情况；证实具有较好的包载效果。第二部分研究分离培养大鼠原代海马神经元，构建环噻唑诱导的细胞癫痫模型，使用Kir2.3转染过表达质粒或特异性开放剂分别上调Kir2.3通道的表达及功能，结果表明Kir2.3通道蛋白激活能够显著抑制海马神经元痫样放电、并对模型小鼠痫性发作及脑电活动具有抑制效果；同时发现其在急性期兴奋活动增强及慢性期病理改变形成的过程中具有神经保护作用。第三部分研究，检测PAMAM-PEG-Angiopep2转染后Kir2.3蛋白表达，进一步验证该质粒作为KCNJ4传输载体在小鼠体内的转染情况，发现右侧海马区域Kir2.3蛋白的表达显著增高；另外还成功检测到致痫模型中KCNJ4与下游共轭分子Caveolin-1的蛋白表达水平均低于对照组，证明两者具有共表达趋势。 . 上述结果均证实内向整流钾通道在难治性癫痫发病机制中具有相关性，提示Kir2.3通道可以作为未来潜在AEDS药物的新型开发靶点。