裸露DNA经胆道分支导入活体肝脏的局部表达

目的 经胆道分支逆行导入裸露DNA,观察目的基因在正常及急性损害大鼠局部肝脏的定量和定位表达.方法 运用D-氨基半乳糖制作大鼠急性肝损害模型;经胆管分支将Cy3荧光标记CMV β质粒通过胆道分支逆行导入局部肝脏,观察裸露DNA导入肝脏的情况.同时运用包含萤火虫荧光素基因质粒pGL3及包含大肠埃希氏菌β半乳糖苷酶基因质粒pCMV β逆行导入大鼠局部肝脏,定量检测荧光素酶和定位检测β半乳糖苷酶在肝脏的表达.应用非参数Kruskal-Wallis法秩和检验进行统计分析.结果 肝组织冰冻切片观察到Cy3荧光标记DNA在汇管区附近聚集.按100 μg/ml浓度进行基因导入,荧光素酶基因在正常大鼠肝脏,急性损害大鼠肝脏均有显著表达.大肠埃希氏菌β半乳糖苷酶定位检测结果显示正常大鼠肝脏及急性肝损害大鼠肝脏内有β半乳糖苷酶显著表达,并可见较多肝细胞表达β半乳糖苷酶. 结论 裸露DNA经胆道逆行导入正常及急性损害肝脏局部能获得较好表达,且无明显加重肝脏损害,该项方法为肝脏基因治疗的实验研究提供了良好的技术平台.

Study on gene delivery via the bile duct to rat liver using naked DNA particles

Objective To evaluate the efficacy of gene delivery to the right lateral lobe of the rat liver via a branch of the bile duct using naked DNA. Methods We evaluated regional gene delivery to the right lateral lobe of the rat liver via a branch of the bile duct, using naked DNA, including pGL3, pCMV β and Cy3 labeled CMV β. Results Gene expression was observed in right lateral lobe of both the damaged and the normal rats liver. The gene delivery efficiency was similar in two groups (P > 0.05). Gene expression was found in the right lateral lobe of damaged and normal livers. Fluorescence was observed in the region of the portal triads, and occasionally, in the lobule. Conclusion Retrograde infusion of naked DNA via the bile duct is an effective way to deliver genes to in both damaged and normal rat liver.