Basic Research for HGF Gene Transduction to Kidney

用HVJ-脂质体诱导HGF基因表达。选择肌肉注射、输尿管内注射或动脉注射作为给药途径，以阴离子或人工病毒膜(AVE)为脂质体，以pUC SRα-HGF、EBNA-HGF或pcDNA3-HGF为载体，寻找最有效的注射途径。实验动物为5周龄Wistar大鼠。双侧股骨肌注4~6次。夹闭肾动静脉后输尿管内注射，5min后结扎输尿管。夹闭肾动、静脉后经肾动脉注入，5min后取下夹子。注射后3d，用抗人HGF抗体用酶联免疫吸附试验检测HGF的表达。HVJ-脂质体外层有一层与HVJ病毒结合的脂膜，其中含有诱导基因。当它与细胞融合时，基因就被引入细胞中。在几种脂质体中，阴离子脂质体和AVE是最常用的。阴离子脂质体是带负电荷的，通常用于体内基因诱导。最近开发的AVE类似于病毒膜。使用了三种启动子不同的载体。结果，输尿管内或动脉内治疗的对侧肾脏HGF的表达没有显著差异。肌内注射法有望避免手术对肾脏损伤的影响。以pcDNA3-HGF为载体的AVE脂质体对HGF基因的诱导效果最好。HGF在肾脏损害中的作用将在以后的研究中按此程序进行。

HGF gene induction using HVJ-liposome was examined. To look for the most efficient method, intra-muscular, intra-ureteral or intra-arterial injection were tested as an administration route, anionic or AVE (artificial viral envelope) were tested in liposome type, and PUC SR α-HGF, EBNA-HGF or pcDNA3-HGF were tested as a vector. Animals used were 5 week-old Wister rat. Intra-muscular injection was made into bilateral femur 4-6 times. Intra-ureteral injection was done after clamping the renal artery and vein and the ureter was ligated 5 minutes later. Intra-arterial injection was done via renal artery after clamping the renal artery and vein and the clamp was removed 5 minutes later. HGF expression was evaluated by ELISA using anti-human HGF antibody 3 days after the injection. HVJ-liposome has a lipid membrane associated with HVJ virus on outer layer and includes induction gene in it. When it fuses with a cell, the gene is introduced into the cell. Among the several kinds of liposome, anionic liposome and AVE were employed. Anionic liposome is negatively charged and is ordinarily used for gene induction in vivo. AVE, which has been developed recently, resembles the viral membrane. Three kinds of vector were used which have different promoters. As a result, there was no significant difference in HGF expression between treated and contralateral kidney by intra-ureteral or intra-arterial. Intra-muscular method was expected to avoid the influence of the surgical damages to the kidney. Finally, HGF gene induction was most efficient when AVE liposome was used with pcDNA3-HGF as a vector. The effect of HGF on renal damages will be investigated according to this procedure in the future.