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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 PSD-95 domain PDZ1 and adenovirus recombinant and observe its effect on kainate (KA) -induced hippocampal neuron apoptosis. Methods The cDNA of PDZ1 was obtained by RT-PCR, ligated with adenovirus shuttle vector pAdTrack-CMV by T4 DNA ligase, and co-transfected with the backbone vector pAdEasy-1 into prokaryotic packaging cell BJ5183. The cells were transformed into eukaryotic packaging cells 293H. The packaged virus particles were purified and their titers were purified. Adenovirus was used to infect hippocampal neurons cultured in vitro. KA was added to the cells and stained with DAPI. Fluorescence microscopy was used to observe apoptosis. Flow cytometry Analyze the rate of apoptosis. Results ① The cDNA of PDZ1 was obtained by RT-PCR. ② The prokaryotic recombinant was identified by electrophoresis. ③ The packaged infectious virus particles were obtained. ④ The titer of purified virus particles was 1.08 × 109ifu / mL. KA induced a decrease in the number of apoptotic hippocampal neurons (P <0.05). Conclusion PDZ1-GFP-expressing virus particles were obtained, and overexpression of PDZ1 antagonized KA-induced apoptosis of hippocampal neurons.