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慢病毒介导shRNA沉默LAMP-2A和TSG101表达对PC12细胞中EGFP-α-synuclein(A53T)降解的影响

, PP. 1393-1399

Keywords: 溶酶体相关膜蛋白-A,肿瘤易感基因,慢病毒,&alpha,-突触蛋白,PC细胞

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Abstract:

目的采用慢病毒介导的shRNA干扰技术,下调PC12细胞中溶酶体相关膜蛋白-2A(lysosome-associatedmembraneproteintype2A,LAMP-2A)和肿瘤易感基因101(tumorsusceptibilitygene101,TSG101)的表达,并研究其对EGFP-α-synuclein(A53T)降解的影响。方法以LAMP-2A和TSG101为靶基因,合成寡核苷酸,退火形成双链DNA,与经BamHⅠ、EcoRⅠ双酶切的pHBLV-U6-Puro载体连接获得重组慢病毒载体;将其与病毒包装辅助质粒共感染293T细胞,收集并浓缩上清液获得重组病毒,测定病毒滴度;病毒颗粒转染PC12细胞,经嘌呤酶素筛选获得稳定感染细胞株;Westernblot检测LAMP-2A和TSG101蛋白的表达;分别采用LAMP-2AshRNA2和TSG101shRNA1下调PC12细胞中LAMP-2A和TSG101蛋白的表达,并结合巴佛洛霉素A1处理,研究其对PC12细胞中EGFP-α-synuclein(A53T)、EGFP、α-synuclein(A53T)和LC3B蛋白的影响,免疫荧光检测各组PC12细胞中EGFP-α-synuclein(A53T)和LC3B蛋白的表达。结果成功构建干扰LAMP-2A和TSG101表达的慢病毒载体,并在293T细胞中包装获得病毒。重组病毒感染PC12细胞后,Westernblot检测结果显示LAMP-2AshRNA2和TSG101shRNA1干扰效果最好;下调LAMP-2A和TSG101蛋白表达均能够抑制细胞中EGFP-α-synuclein(A53T)、EGFP和α-synuclein(A53T)蛋白的降解(P<0.01),并提高LC3-Ⅱ蛋白的含量(P<0.01)。结论慢病毒介导的shRNA能有效地沉默PC12细胞中LAMP-2A和TSG101的表达,抑制细胞中EGFP-α-synuclein(A53T)的降解。

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