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目的观察持续活化突变体腺病毒转染大鼠心肌细胞的转染效果及对乙醛脱氢酶2(ALDH2)表达的影响。方法分别将扩增得到的ALDH2持续活化突变体基因及合成ALDH2-siRNA序列颈环状DNA,连接相应载体后,得到重组穿梭质粒;对2种穿梭质粒分别进行扩增和酶切鉴定,并导入pAdeno腺病毒载体,转染293细胞进行扩增与纯化。将1日龄雄性SD大鼠心肌细胞进行培养,将2种重组腺病毒及对照空载体分别感染细胞,随后检测ALDH2表达量。结果 2种载体构建正确,纯化后两者滴度分别为2×1010,1.6×1010PFU·mL-1。实验组ALDH2表达量与对照组相比差异具有统计学意义(P<0.01)。结论成功构建大鼠ALDH2基因双向调控腺病毒载体,可以有效调控离体大鼠心肌细胞ALDH2表达。
Objective To observe the transfection efficiency and the effect of ALDH2 on the transfection of rat cardiac myocytes transduced with the sustained-activated mutant adenovirus. Methods The ALDH2 sustained-activating mutant gene and the synthetic circular DNA of ALDH2-siRNA sequence were synthesized and ligated with the corresponding vector respectively to obtain a recombinant shuttle plasmid. The two shuttle plasmids were respectively amplified and digested with restriction endonucleases pAdeno adenovirus vector, transfected 293 cells for amplification and purification. One-day-old male SD rat cardiomyocytes were cultured, and two kinds of recombinant adenovirus and control empty vector were infected respectively, and then ALDH2 expression was detected. Results The two vectors were correctly constructed and their titers were 2 × 1010 and 1.6 × 1010 PFU · mL-1, respectively. The experimental group ALDH2 expression compared with the control group, the difference was statistically significant (P <0.01). Conclusion The successful construction of rat ALDH2 gene bidirectional regulatory adenovirus vector can effectively regulate the ALDH2 expression in isolated rat cardiomyocytes.