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为了检测REST蛋白过表达或干扰对由PrP106-126毒性多肽引起的原代神经元纤维的影响,本试验通过免疫荧光技术和免疫印迹方法分别检测受PrP106-126刺激后的原代神经元中REST表达的变化情况。通过脂质体转染方法将已经构建好的pCMV-HA-REST质粒转染进入原代神经元,或利用靶向REST的小干扰siRNA技术干扰原代神经元中REST蛋白的表达。用PrP106-126毒性多肽刺激转染了REST过表达或干扰质粒的原代神经元,利用免疫印迹检测突触后蛋白PSD-95的变化情况;利用免疫荧光观察突触后蛋白和神经纤维的损伤情况。结果显示,受到多肽刺激后,REST从神经元的胞浆向胞核转移的同时表达量也逐渐增加,24h时达到顶峰,之后缓慢减少。生理情况下,过表达REST促进突触后蛋白PSD-95的表达;病理情况下,REST的过表达减轻由毒性多肽引起的突触损伤、神经纤维断裂。相应地,干扰REST后,加剧毒性多肽对神经纤维的损伤。结果表明,REST蛋白的过表达缓解PrP106-126毒性多肽对原代神经细胞造成的病理性损伤,为进一步阐释REST对朊病毒及相关神经退行性疾病引起的病理性损伤的治疗作用和神经保护机制提供了理论依据。
In order to detect the effect of REST protein overexpression or interference on primary neuronal fibers induced by PrP106-126 cytotoxicity, we tested whether the REST protein expression in primary neurons stimulated by PrP106-126 was detected by immunofluorescence and Western blotting Changes in expression. The constructed pCMV-HA-REST plasmids were transfected into primary neurons by liposome transfection method, or the expression of REST protein in primary neurons was interfered by REST siRNA targeting small interfering siRNA. Primary neurons transfected with REST over-expression or interference plasmids were stimulated with PrP106-126 toxic polypeptide and the changes of postsynaptic protein PSD-95 were detected by immunoblotting. The damage of postsynaptic proteins and nerve fibers was observed by immunofluorescence Happening. The results showed that, after being stimulated by the polypeptide, REST also increased gradually from the cytoplasm to the nucleus of neurons, and reached the peak at 24 hours, then slowly decreased. Under physiological conditions, overexpression of REST promotes the expression of PSD-95; in pathological conditions, overexpression of REST alleviates synaptic damage and nerve fiber rupture caused by toxic peptides. Correspondingly, the interference of REST aggravates the injury of nerve fibers by toxic peptides. The results showed that overexpression of REST alleviates the pathological damage caused by PrP106-126 to primary neurons. To further elucidate the therapeutic effect and mechanism of REST on pathological damage caused by prion and related neurodegenerative diseases Provided a theoretical basis.