siRNA干扰SIAH对细胞活性和α―synuclein泛素化降解通路影响.docVIP

siRNA干扰SIAH对细胞活性和α―synuclein泛素化降解通路影响 　　【摘要】 目的：研究siRNA干扰SIAH基因对神经母细胞瘤细胞SH-SY5Y细胞α-synuclein泛素化、降解通路的影响。方法：用荧光标记的siRNA-FAM转染SH-SY5Y细胞后，专业设计合成3条针对SIAH的siRNA，Western Blot检测蛋白水平SIAH表达的变化，筛选出其中最高效的1条siRNA，用CCK-8法检测该siRNA-SIAH干扰SH-SY5Y细胞活性；流式细胞技术检测该siRNA-SIAH干扰SH-SY5Y细胞凋亡的影响；Western Blot检测α-synuclein、LC3、E1、P53蛋白水平表达的变化；qRT-PCR检测SIAH、α-synuclein、LC3、E1 mRNA水平表达的变化；免疫共聚焦分别检测SIAH、α-synuclein、LC3共定位情况。结果：3条siRNA均成功转染SH-SY5Y细胞，流式细胞术检测siRNA转染SH-SY5Y细胞转染率89%，Western结果显示其中siRNA-2#使细胞中SIAH蛋白表达明显降低，选取该序列进行研究。CCK-8法结果显示该siRNA干扰SIAH后对SH-SY5Y细胞活增高，流式细胞技术检测结果显示该siRNA干扰SIAH可抑制SH-SY5Y细胞的凋亡，与空白对照组、阴性对照组比较，差异均有统计学意义（P0.05）。通过Western Blot检测siRNA-2#组的α-synuclein、LC3、P53蛋白水平，与空白对照组、阴性对照组比较均明显降低，E1蛋白水平增高，差异均有统计学意义（P0.05）。通过qRT-PCR检测siRNA-2#组的SIAH、α-synuclein、LC3-Ⅱ mRNA水平与空白对照组、阴性对照组比较均明显降低，而E1的mRNA水平明显升高，差异均有统计学意义（P0.05）。结论：应用siRNA干扰SIAH基因，通过促进泛素-蛋白酶体系统作用减少SH-SY5Y中α-synuclein聚集，SIAH基因有可能成为帕金森病治疗中的一个新靶点。 　　【关键词】 帕金森病； E3泛素连接酶； α-synuclein； 泛素化蛋白酶体 　　【Abstract】 Objective：To study the effect of siRNA-SIAH on α-synuclein autophagy and UPS degradation in SH-SY5Y.Method：After transfection of siRNA-FAM cells with fluorescent labeled SH-SY5Y， 3 siRNA were professional designed for SIAH，the protein level of SIAH expression was measured by Western Blot， one of the most efficient siRNA was selected.CCK-8 assay was used to detect the activity of siRNA-SIAH in SH-SY5Y cells. Flow cytometry was used to detect the apoptosis of siRNA-SIAH in SH-SY5Y cells.Western Blot was used to detect the protein level expression ofα-synuclein，LC3，E1，and P53.qRT-PCR was used to detect the level of mRNA in SIAH，α-synuclein，LC3 and E1. Confocal microscopy was used to detect the positioning of SIAH，α-synuclein and LC3-Ⅱ.Result： 3 siRNA cells successfully transfected SH-SY5Y，the transfection efficiency was 89%. The Western results showed that the expression of SIAH was significantly decreased in siRNA-2# cells， then studied this sequence. CCK-8 assay showed that the SH-SY5Y cell activity increased after siRNA interference