慢病毒介导APP695基因RNAi对体外培养的APP695转基因小鼠皮质神经元β淀粉样蛋白分泌的影响  

Effect of lentivirus-mediated APP695 RNAi on Aβ level in cortical neurons of APP695 transgenic mice

作　　者：赵莘瑜[1] 温丽君[1] 李国栋[2] 韩志强[1] 宋波[1] 许予明[1] 

机构地区：[1]郑州大学第一附属医院神经内科,450052 [2]郑州大学医学实验中心

出　　处：《中华医学杂志》2010年第45期3225-3230,共6页National Medical Journal of China

基　　金：基金项目：河南省教育厅自然科学研究计划（2009A320045）;河南省卫生厅重点项目（200702005）;河南省科技厅杰出青年基金（74100510001）

摘　　要：目的 探讨慢病毒介导RNA干扰（RNAi）抑制APP695基因表达对体外培养的APP695转基因小鼠皮质神经元细胞分泌β淀粉样蛋白（Aβ）的影响.方法 构建靶向APP695基因的短发夹状RNA（shRNA）慢病毒表达质粒（pFU-GW-iRNA）,进行酶切和测序鉴定.慢病毒表达质粒与包装质粒（pHelper 1.0和Helper 2.0）共转染293T细胞,获得病毒浓缩液并测定滴度.使用APP695-shRNA慢病毒载体感染体外培养的APP695转基因小鼠皮质神经元,设定为慢病毒介导阳性干扰（APP695-RNAi）组,另设阴性对照病毒感染（NC）组、未经病毒感染（CON）组.采用实时荧光定量PCR检测APP695基因mRNA的表达,Western印迹检测APP695蛋白的表达,采用Elisa检测Aβ40和Aβ42的生成.结果 PCR扩增和测序结果证实,APP695 shRNA核苷酸链序列插入正确,包装慢病毒产生病毒悬液的滴度为5×108 TU/ml.使用慢病毒载体感染体外培养的APP695转基因小鼠皮质神经元,实时荧光定量PCR检测APP695-RNAi组APP695基因mRNA的抑制率为76.70%,与NC组和CON组相比差异有统计学意义（P＜0.001）.Western印迹结果显示蛋白水平表达下降与定量PCR一致.Elisa检测干扰72 h后APP695-RNAi组、NC组、CON组Aβ40的分泌分别为（184±15）ng/L、（647±30）ng/L、（656±40）ng/L.APP695-RNAi组与NC组和CON比较差异均有统计学意义（均为P＜0.001） APP695-RNAi组、NC组、CON组Aβ42的分泌分别为（19.2±1.9）ng/L、（67.6±6.0）ng/L、（68.6±7.0）ng/L.APP695-RNAi组与NC组和CON比较差异均有统计学意义（均为P＜0.001）.结论 慢病毒载体介导的APP695基因RNA干扰可以有效抑制痴呆小鼠皮质神经元细胞Aβ40和Aβ42的分泌.Objective To explore the effect of lentivirus-mediated APP695 RNAi on Aβ level in the cortical neurons of APP695 transgenic mice.Methods The pFU-GW-iRNA plasmids expressing the RNAi sequences of human APP695 gene were confirmed by PCR （polymerase chain reaction） and sequencing.The recombined plasmids in combination with pHelper 1.0 and Helper 2.0 plasmids were transfected into 293T cells so as to construct the lentiviral vectors of RNA interference of APP695 gene.Virus in supernatant was collected and the viral titer measured.The cortical neurons of APP695 transgenic mice were infected with the packaged lentivirus.And it was designated as APP695-RNAi group.Cells infected with APP695-NC was designated as NC group and non-infected cells as CON group.The levels of APP695 mRNA were detected by real-time quantitative PCR.The APP695 protein was detected by Western blot and the levels of Aβ40 and Aβ402 were measured by ELISA （enzyme-linked immunosorbent assay）.Results PCR and DNA sequencing demonstrated that the inserted sequences of APP695 shRNA were correct.The viral titer was 5 ×108 TU/ml.After the infection of APP695 shRNA recombinant lentivirus,the expression level of APP695mRNA in the cortical neurons of APP695 transgenic mice decreased significantly （inhibition rate：76.70%）versus NC and CON groups（P 〈0.001）.The lowered expression of APP695 proteins was consistent with APP695 mRNA.The levels of Aβ40 were （184 ± 15） ng/L,（647 ±30） ng/L and （656 ±40） ng/L in APP695-RNAi,NC and CON groups respectively.As compared with NC and CON groups,the levels of Aβ40 in APP695-RNAi group decreased significantly（P 〈0.001）.The levels of Aβ42 were （19.2 ± 1.9）ng/L,（67.6 ± 6.0） ng/L and （68.6 ± 7.0） ng/L in APP695-RNAi,NC and CON groups respectively.As compared with NC and CON groups,the levels of Aβ42 decreased significantly in APP695-RNAi group （P 〈0.001）.Conclusion The inhibition of APP695 by lentivirus-mediated RNAi effectvely decreases the levels of Aβ40 and

关 键 词：基因 RNA干扰 慢病毒属 小鼠 转基因 

分 类 号：R686[医药卫生—骨科学]