重组Nkx2．5腺病毒的构建及心肌细胞感染实验

利用AdEasy腺病毒表达系统构建了Nkx2．5重组腺病毒．Nkx2．5基因克隆入pAdTrackcmv质粒，pAdTtrackcmv—Nkx2．5经Pme I线性化后转化含pAdEasy-1的BJ5183菌进行同源重组，以构建pAdEsay-Nkx2．5重组质粒．pAdEsay—Nkx2．5转染293A细胞进行病毒包装．以Ad-Nkx2．5重组腺病毒感染Hela及乳鼠心肌细胞，检测了Nkx2．5蛋白表达及对下游ANF和β-MHC基因的表达调控；并采用感染病毒的H9c2心肌细胞经H2O2处理建立细胞凋亡模型，采用噻唑蓝法和荧光染色法检测了细胞存活率和凋亡细胞的形态变化．结果显示，Nkx2．5过表达能抑制H2O2诱导的H9c2细胞凋亡．

Construction of recombinant adenovirus Nkx2.5 and its infection in myocardial cells

The AdEasy system was used to create recombinant adenovirus expressing Nkx2. 5 gene. Nkx2. 5 gene was cloned into pAdTrackcmv. The pAdTrackcmv-Nkx2.5 plasmid was linearized by Pme I digestion, and then transformed into E. coli BJ5183 containing pAdEasy-1. The pAdEsay-Nkx2.5 plasmid was transfected into 293A packaging cells to produce recombinant adenovirus Ad-Nkx2.5. Hela cells and neonatal rat cardiac myocytes were infected with adenovirus Ad-Nkx2.5 to assess Nkx2.5 expression and its function on regulating downstream genes, such as ANF and β-MHC. H9c2 myocardial cells after infection with adenovirus were subjected to H2O2 to induce apoptosis. MTT assay and fluorescence staining were performed to evaluate cells surival rate and cellular morphology of H9c2 cells. The results indicated that Nkx2. 5 overexpression could protect H9c2 cells from apoptosis.