In vivo HGF gene transfer promoted liver regeneration and inhibited lethal liver failure after massive hepatectomy in rats with cirrhosis

背景/目的：肝手术被认为是治疗肝细胞癌的潜在治疗方法之一。然而，大多数患者同时存在肝硬变和肝脏储备功能受损。因此，大范围肝切除往往会导致肝硬变患者术后发生致死性肝功能衰竭。肝细胞生长因子(HGF)是一种强有力的肝细胞有丝分裂原，在肝再生中起重要作用。本研究将HGF基因导入肝硬变大鼠肝脏，观察HGF基因转导对肝切除后致死性肝功能衰竭的预防作用。方法：1%二甲基亚硝胺(DMN)诱导大鼠肝纤维化，每周连续3天，连续4周。最后一次注射DMN后3d，用日本血凝素-脂质体将携带巨细胞病毒启动子的20μg肝细胞生长因子基因经门静脉注入肝脏。对照组大鼠按相同方法注射**S注射液。三分之二的肝切除…根据Higgins和Anderson描述的过程，更多的y在注射HGF基因或PBS后3天*形成。于2/3肝切除后6、12、24、72、168h处死大鼠，采集血样和肝组织。用增殖细胞核抗原免疫组织化学方法检测肝细胞DNA合成。采用原位末端标记法检测肝细胞凋亡率。用Western blotting分析促凋亡和抗凋亡蛋白Bax和Bclxl的表达。结果：对照组大鼠在肝切除后6h开始死亡，3d后死亡31.5%。另一方面，HGF基因转导显著提高了死亡率，7d存活率达92.7%。肝切除3d后，用增殖细胞核抗原检测转HGF基因大鼠肝细胞DNA合成高于对照大鼠肝细胞DNA合成，但肝残肝体积两组间无显着性差异。原位末端标记法显示，对照组大鼠肝切除后早期可见大量肝细胞凋亡，而肝细胞生长因子基因转染组仅见少量肝细胞凋亡。尽管在肝切除术后早期，抗凋亡蛋白Bclxl在正常肝脏中的表达显著增加，但在本研究中，这些蛋白在肝硬变大鼠的肝脏中被显著抑制。肝细胞生长因子基因转移到肝硬变后可显著改善肝切除后这些蛋白的表达。另一方面，促凋亡蛋白Bax在正常大鼠肝切除后3天未见表达，而在对照组和转导HGF的大鼠肝切除后已有表达。结论：肝硬变大鼠部分切除后，肝细胞生长因子基因治疗有效地阻止了肝细胞的凋亡，提示肝细胞生长因子可能是作为一种抗凋亡因子而不是促再生因子发挥作用。HGF的这种作用可能在一定程度上抑制了肝部分切除后早期的死亡率。较少

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 … More 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