OKAMURA Haruki Hyogo College of Medicine, Medical school, Professor, 医学部, 教授 (60111043)
TAKEUCHI Masaharu Hyogo College of Medicine, Medical school, Research Associate, 医学部, 助手 (00258162)
FUJIMOTO Jiro Hyogo College of Medicine, Medical school, Professor, 医学部, 教授 (90199373)
|Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
Background/Aim : Liver surgery is accepted as one of the potentially curative treatments for hepatocellular carcinoma. However, most patients have coexisting cirrhosis and impaired liver reserve function. Therefore, massive hepatectomy often causes lethal liver failure post operation in cirrhotic liver. Hepatocyte growth factor (HGF) is known to be a potent mitogen in hepatocyte and play an important roll in liver regeneration. In the present study, we delivered HGF gene to cirrhotic rat liver, and investigated whether HGF gene transduction prevents lethal liver failure after hepatectomy.
Method : Liver cirrhosis was induced by administrating 1% dimethylnitrosamine (DMN) on 3 consecutive days a week for 4 weeks. Three days after the final injection of DMN, 20 μg of HGF gene with CMV promoter was introduced to liver using hemagglutinating virus of Japan (HVJ)-liposome via portal vein with clamp of left glisson. Control rats received **S injection in the same method. Two-thirds hepatectom
y, according to the procedure described by Higgins and Anderson, was ***formed 3 days after HGF gene or PBS injection. On hours 6,12,24,72,168 after the two-thirds hepatectomy, rate were sacrificed and the blood sample and liver tissue were collected. DNA synthesis in hepatocytes was investigated with immunohistochemistry of proliferating cell nuclear antigen (PCNA). Apoptotic hepatocytes were determined by using the TUNEL assay. The expression of pro-apoptotic and anti-apoptotic protein, such as Bax and Bcl-xL, was analyzed by Western blotting.
Results : In the control group, rat began to die 6 hours after hepatectomy, and 31.5% of rats died by 3 days. On the other hand, HGF gene transduction significantly improved mortality, and 92.7% of rats survived at 7days. DNA synthesis of hepatocytes in the HGF gene transferred rat liver determined with PCNA was higher than those in the control rat liver 3 days after hepatectomy while no significant difference in the remnant liver volume was observed between the groups. A number of apoptotic liver cells were observed with TUNEL method on early time course after the hepatectomy in the control group, whereas few apoptotic figures were detected in the HGF gene transferred liver. Though expression of anti-apoptotic proteins, Bcl-xL, has been shown to increase substantially in normal liver at early phase after hepatectomy, these proteins were remarkably inhibited in the cirrhotic rat liver in this study. HGF gene transfer into the cirrhotic livers significantly improved the expression of these proteins after hepatectomy. On the other hand, expression of pro-apoptotic protein Bax was not seen in normal rat liver by 3 days after hepatectomy, but this protein has been already expressed following hepatectomy in the control and HGF transducted rats. HGF gene transduction was not seen obvious effects against Bax protein in cirrhotic rats.
Conclusion : After partial resection of cirrhotic liver, HGF gene therapy effectively prevented apoptosis of hepatocyte, suggesting that HGF worked as an anti-apoptotic agent rather than a regeneration-accelerating factor. This effect of HGF possibly inhibited the mortality in early time course after partial hepatectomy. Less