表达大鼠caspase-3 siRNA腺病毒的构建及其对神经元凋亡的影响

Construction of adenovirus expressing caspase-3 siRNA and its effect on neuronal apoptosis

目的构建表达大鼠caspase-3 siRNA腺病毒，体外观察其抑制内毒素诱导神经元凋亡的效果。方法合成正、反义寡核苷酸并克隆入pShuttleH1载体，与含caspase-3及绿色荧光增强蛋白序列的质粒pEGFPC1-Cas3共转染293细胞，流式细胞仪分析荧光强度；pShuttleH1-siCas3线性化后转化到含pAdEasy-1的BJ5183感受态细菌中获取重组质粒，脂质体转染293细胞获取重组腺病毒Ad-siCas3；TCID50法测定病毒滴度；感染海马神经元，观察内毒素诱导神经元caspase-3 mRNA的表达及凋亡情况。结果目的基因序列正确，pShuttleH1-siCas3可明显减弱pEGFPC1-Cas3绿色荧光，Ad-siCas3滴度为1．06×10^10pfu／ml。转染病毒的海马神经元内毒素诱导的凋亡效应及caspase-3 mRNA的表达明显减弱。结论成功地构建了表达大鼠caspase-3 siRNA腺病毒，具有良好的抑制神经元凋亡作用。

Objective To construct the incompetent-repllcation adenovirus expressing rat caspase-3 siRNA and to identify its effect on LPS-induced neuronal apoptosis in vitro. Methods The complementory DNA containing both sense and antisense oligo DNA of the targeting sequence was cloned into the pShuttle H1 vector. The 293 cells which contained caspase-3 and EGFP sequences were co-transfected with pShuttle Hl-siCas 3 and pEGFPC1-Cas 3 which contained caspase-3 and EGFP sequences. The linearized pShuttle Hl-siCas 3 was transformed into E coli BJ5183 cells containing backbone plasmid pAdEasy-1 by electroporation. The 293 cells were transfected with the recombinant plasmid to package the adenovirus Ad-siCas 3. The titers of adenovirus were determined using the specific 50% tissue culture infection dosage （TCID50） method. After the cultured hippocampal neurons were infected by virus, LPS-induced apoptosis and caspase-3 mRNA expression in hippocampal neurons were observed. Results It was identified that the sequence of fusion gene was correctly inserted into the genome of virus, pShuttleHl-siCas3 reduced the expression of green fluorescence protein in 293 cells. The titer of recombinant adenovirus was 1.06 ×10^10 pfu/ml. Neuronal apoptosis and caspase-3 mRNA were greatly reduced after virus infection. Conclusion The recombinant adenovirus expressing rat caspase-3 siRNA were successfully constructed for the first time and can be used in pain therapy by its anti-apoptosis effect.