Cre／LoxP位点特异性重组系统构建Ad．CMV-热休克蛋白70腺病毒载体及其对肝细胞缺血再灌注损伤的保护作用

Construction of Ad. CMV-HSP70 and its protective effect on the ischemla-reperfusion injury in liver cells in vitro

目的探讨重组腺病毒介导的热休克蛋白70（HSP70）基因转染对肝细胞缺血再灌注损伤的保护作用。方法用Cre酶介导的loxP位点特异性重组的方法，构建HSP70腺病毒载体；体外转染人正常肝细胞株，检测HSP70的表达水平；对照组（转染空载体组）、转染HSP7024、48、72h组细胞经缺氧再复氧处理后，分别对细胞的存活率进行检测分析。结果重组产物经鉴定后为包含有HSP70全长序列的腺病毒载体，HSP70基因位于其CMV启动子控制之下，浓度为3．0×10^10pfu／ml。共2ml。Ad．CMV，HSP70转染组细胞HSP70基因表达为阳性，对照组无表达；经缺氧再复氧处理后，Ad．CMV—HSP70处理组活力较对照组明显增强，死亡细胞明显减少。结论Cre酶介导的loxP位点特异性重组是一种简单、高效的腺病毒载体构建方法，具有构建速度快、成功率高、准确率高等特点。

Objective To explore the protective effects of recombinant adenovirus-mediated HSP70 gene transfection on the ischemia-repeffusion injury of liver cells. Methods Recombinant adenovirus vector carrying HSP70 was constructed with full length human HSP70 gent by Cre-mediated site-specific recombination method. After identified, the desired Ad vectors were purified by density gradient ultracentrifuge and titrated. Cultured liver cells were divided into 4 group： the control group was transfected with vacant recombinant adenovirus, three group were transfected with Ad. CMV-HSP70 for 24,48,72 h respectively. The expression of tranefectcd HSP70 was detected by RT-PCR after transfection. After the cells in the four groups suffered 4 hours of hypoxia followed by 8 hours of reoxygenation, the cell viability was analyzed by MTr method. Results The replication-deficient adenovirus vectors coding human HSP70 DNA were generated in high titer which roached 3.0 × 10^10 pfu/ml after density gradient ultracentrifuge. Compared with the control group,the expression of HSP70 were obviously increased after transfection,and the peak level was at 48 h after transfection. After hypoxia-reoxygenation, the cell viability rates of HSP70 gene transfected groups were （70.8 ± 5.3 ） %, （ 89.4 ± 5.2） %, （ 80.1 ± 5.7） % respectively, all significantly higher than that of control group （ P 〈 0.01 ）. Conclusion The efficiency and reliability of this Cre-mediated slte-specific recombination method greatly simplify and expedite the construction of recombinant Ad vectors containing the human HSP70. The over expression of human HSP70 mediated by recombinant adenovirus can protects liver cell against hypoxia-reoxygenation injure in vitro.