摘要
目的构建PSD-95结构域PDZ1与腺病毒重组体并观察其对海人藻酸(KA)诱导的海马神经元凋亡的作用。方法采用RT-PCR法获得PDZ1的cDNA;与腺病毒穿梭载体pAdTrack-CMV用T4 DNA连接酶连接;连接产物与骨架载体pAdEasy-1共转染至原核包装细胞BJ5183;将原核重组体用脂质体转入真核包装细胞293H;纯化包装后的病毒颗粒并测定其滴度;腺病毒感染培养的海马神经元,加入KA,用DAPI染色后荧光显微镜观察细胞凋亡;用流式细胞仪定量分析细胞凋亡率。结果①经RT-PCR得到了PDZ1的cDNA;②电泳鉴定得到原核重组体;③得到包装完整的具感染能力的病毒颗粒;④纯化的病毒颗粒滴度为1.08×109ifu/mL⑤病毒表达的PDZ1使KA诱导的海马神经元凋亡数量减少(P<0.05)。结论获得了能表达PDZ1-GFP的病毒颗粒,并且PDZ1过表达能拮抗KA诱导的海马神经元凋亡。
Objective To construct recombinant of Ad-PDZ1-GFP and detect its effect on apoptosis in neurons induced by kainic acid （KA）. Methods Using the total RNA as template, the cDNA of PDZ1 was amplified by RT-PCR; the cDNA was subsequently cloned into the shuttle vector pAdTrack-CMV; the resultant plasmid is co-transformed into E. coli BJ5183 cells with an adenoviral backbone plasmid pAdEasy- 1; the recombinant plasmid is transfected into adenovirus packaging cell lines HEK 293 cells with Lipofectamine; the particles of Ad-PDZ1-GFP was purified and tittered; the neurons infected with the viruses were treated with kainic acid and then were incubated with DAPI for characterizing apoptosis-like cells using fluorescence; quantification of the apoptotic rate was examined by FCM. Results （1）The cDNA of PDZ1 was amplified by RT-PCR. （2）The procaryon recombination was confirmed by restriction endonuclease analyses. （3） Recombinant adenoviruses were typically generated. （4） Titer of adenoviruses was 1.08 × 10^9 ifu/mL. （5） Apoptosis was obviously decreased in neurons expressing PDZ1 （P 〈 0. 05）. Conclusion Ad-PDZ1-GFP particles are obtained successfully, which can express protein PDZ1-GFP and the overexpression of PDZ1 rescues hippocampl neurons from apoptosis induced by kainic acid.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2008年第6期651-655,共5页
Immunological Journal
基金
国家自然科学基金(30330190)
江苏省高校自然科学基础研究基金(07KJB310118)
徐州医学院科研基金(07KJ15)资助课题
