肾脑表达蛋白过表达载体对于β-淀粉样蛋白诱导神经细胞凋亡的作用机制  

The mechanism of KIBRA overexpression vector on Apoptosis of neuronal cells induced by Aβ

作　　者：支海鸯 陈凌[1] 黎斌[1] ZHI Hai-yang;CHEN Ling;LI Bin(Department of Neurology,Wenzhou Hospital of Traditional Chinese Medicine Affiliated to Zhejiang Chinese Medical University,Wenzhou,Zhejiang 325000,China)

机构地区：[1]浙江中医药大学附属温州市中医院神经内科,浙江温州325000

出　　处：《中国卫生检验杂志》2020年第11期1356-1359,共4页Chinese Journal of Health Laboratory Technology

摘　　要：目的探究肾脑表达蛋白(KIBRA)过表达载体对于β-淀粉样蛋白(Aβ)诱导神经细胞凋亡的作用机制。方法本研究以原代海马神经细胞为研究对象,利用20μmol/L Aβ25-35诱导,构建体外稳定的AD细胞模型,利用siRNA技术构建KIBRA过表达载体,由慢病毒载体转染AD细胞模型,按照处理方法将AD细胞模型分成3组,即空白对照组、阴性对照组和过表达载体组。利用RT-PCR和Western blot检测KIBRA、Caspase-3以及Caspase-9的表达水平,利用AV-PI试剂盒检测细胞凋亡。结果 AD细胞模型中,可见细胞核汇聚,细胞形态不规则,触角形成。慢病毒滴度的检测结果显示KIBRA慢病毒过表达载体慢病毒转染滴度为7×106TU/ml。空白对照组、阴性对照组和过表达载体组的KIBRA的mRNA的相对表达量比较差异有统计学意义(F=9. 068,P <0. 05),过表达载体组的KIBRA的mRNA的相对表达量高于空白对照组和阴性对照组(q值分别为2. 021、2. 981,P <0. 05)。过表达载体组的Caspase-3、Caspase-9的mRNA的相对表达量低于空白对照组和阴性对照组(P <0. 05)。Western blot结果与RT-PCR表达趋势相同。AV-PI结果显示,过表达载体组的细胞总凋亡率低于空白对照组和阴性对照组(q值分别为2. 551、3. 003,P <0. 05)。结论 KIBRA的过表达载体可以抑制AD细胞模型的凋亡,可以作为阿尔茨海默病的潜在治疗靶点。Objective To explore the mechanism of KIBRA overexpression vector on neuronal apoptosis induced by Aβ.Methods Primary hippocampal neurons were induced by 20 μmol/L Aβ25-35 to construct stable AD cell model in vitro.KIBRA overexpression vector was constructed by siRNA technology. AD cell model was transfected by lentivirus vector. According to the treatment method,AD cell model was divided into three groups: blank control group,negative control group and overexpression vector group. The levels of KIBRA,Caspase-3 and Caspase-9 were detected by RT-PCR and Western blot.Apoptosis was detected by AV-PI kit. Results In AD cell model,nucleus convergence,irregular cell morphology and antenna formation were observed. The results of lentivirus titer test showed that the transfection titer of KIBRA lentivirus overexpression vector lentivirus was 7 × 106 TU/ml. The relative expression of KIBRA mRNA in blank control group,negative control group and overexpression vector group had statistical difference( F = 9. 068,P < 0. 05). The relative expression of KIBRA in overexpression vector group was significantly higher than that in blank control group and negative control group( q = 2. 021,q =2. 981,P < 0. 05). The relative expressions of Caspase-3 and Caspase-9 in the overexpression vector group were significantly lower than that in the blank control group and the negative control group( P < 0. 05). Western Blot results showed the same expression trend as RT-PCR. AV-PI results showed that the total apoptotic rate of the overexpression vector group was significantly lower than that of the blank control group and the negative control group( q = 2. 551,q = 3. 003,P < 0. 05). Conclusion KIBRA overexpression vector can inhibit the apoptosis of AD cell model and may be a potential therapeutic target for Alzheimer’s disease.

关 键 词：慢病毒 肾脑表达蛋白 阿尔茨海默病 凋亡 

分 类 号：R749.16[医药卫生—神经病学与精神病学]