Beclin1基因慢病毒载体的鉴定包装及其在银杏酮酯保护衰老海马神经元中的作用  

Identification and package of lentiviral vector for Beclin1 gene and the role of it in Ginkgo Biloba extract protecting H_2O_2-induced senescence-like hippocampal neurons

作　　者：郝莉[1] 徐玉英[1] 郭春霞[2] 张志雄[2] 高崎 顾慧芬 

机构地区：[1]河南中医学院基础医学院,郑州450046 [2]上海中医药大学基础医学院,上海201203 [3]上海杏灵科技药业有限公司,上海200127

出　　处：《中药药理与临床》2015年第4期90-94,共5页Pharmacology and Clinics of Chinese Materia Medica

基　　金：上海市科委科研计划项目(09ZR1432100)

摘　　要：目的:探讨自噬相关基因Beclin1在银杏酮酯保护衰老海马神经元中的作用。方法:使用酶切和DNA测序方法对已构建好的p LVX-shRNA2-Beclin1慢病毒载体进行鉴定,并与辅助包装质粒载体共同转入293T细胞得到病毒颗粒。将病毒颗粒转染原代大鼠海马神经元。培养原代大鼠海马神经元并随机分为6组:正常组、模型组、银杏酮酯组、银杏酮酯干扰组、阳性对照银杏叶提取物组和银杏叶提取物干扰组。正常组用正常培养基培养,模型组用H2O2(200μM)诱导18小时建立衰老样细胞模型;银杏酮酯和银杏叶提取物组分别用200μg/ml的药物预处理6小时后再加入H2O2诱导18小时;其余两组在银杏酮酯和银杏叶提取物组基础上分别加入慢病毒载体颗粒。通过荧光定量PCR和免疫印迹方法检测Beclin 1、LC3-Ⅱ和SyntheinⅠ表达。结果:酶切和DNA测序结果表明重组慢病毒载体p LVX-shRNA2-Beclin1的插入序列正确。用293T细胞包装的重组慢病毒能够感染原代大鼠海马神经元。体外荧光定量PCR结果显示:慢病毒颗粒转染衰老海马神经元后,200μg/ml银杏酮酯和银杏叶提取物干扰组Beclin1 mRNA水平降低。免疫印迹结果显示:与模型组相比,200μg/ml银杏酮酯和银杏叶提取物组Beclin1、LC3-Ⅱ和Synpain I蛋白表达均增加;慢病毒颗粒转染后,200μg/ml银杏酮酯和银杏叶提取物干扰组神经元Beclin1、LC3-Ⅱ和Synpain I蛋白表达明显降低。结论:Beclin1与银杏酮酯(200μg/ml)保护衰老海马神经元有关。Objective： To investigate the interfering efficiency of Beclin1 gene in Ginkgo Biloba Extract（ GBE50） protecting in H2O2-induced senescence-like hippocampal neurons. Method： Single digest and DNA sequencing were used to confirm the constructed vectors whether or not success recombinant vector plasmid and to coinfect 293 T cells,and then was transfected into senescence-like primary hippocampal neurons.Primary hippocampal neurons were cultured and the cells were divided into six groups： control group,model group,GBE50 group,GBE50 interfere group,positive control EGB761 group and EGB761 interfere group. Cells treated only with solvent for 24 h were used as control and cells treated with H2O2 for 18 h were used as model. The cells were incubated with GBE50 or EGB761 for 6 h before being treated with H2O2（ 200 μM） for 18 h. The other two groups were added into the lentiviral vector particles on the basis of GBE50 and EGB761 groups. Realtime PCR was used to detect Beclin1,and Western-blot was performed to examine Beclin1,LC3-Ⅱand Synpain I protein levels. Results：Single digestion and DNA sequencing showed that recombinant lentivirus plasmids p LVX-shRNA2-Beclin1 were constructed successfully. The recombinant lentivirus plasmids was transfected into senescence-like hippocampal neurons. In vitro experiment,Real-time PCR confirmed that Beclin1 mRNA was low-expressed in GBE50 and EGB761 interfere groups after the lentiviral particles was transfected into senescencelike hippocampal neurons. Western blot displayed that Beclin1,LC3-Ⅱand Synpain I protein level increased in GBE50 and EGB761 groups,compared to model group. However,Beclin1,LC3-Ⅱand Synpain I protein level were all decreased in GBE50 and EGB761 interfere groups after the lentiviral particles was transfected into neurons. Conclusion： Beclin1 gene was related to Ginkgo Biloba Extract protecting H2O2-induced senescence-like hippocampal neurons.

关 键 词：银杏酮酯 BECLIN1 慢病毒载体 衰老 海马神经元 

分 类 号：R285[医药卫生—中药学]