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

背景/目的：肝外科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化和肝储备功能受损。因此，大面积肝切除术常导致肝硬化术后致死性肝功能衰竭。肝细胞生长因子（HGF）是肝细胞中一种有效的丝裂原，在肝脏再生中起着重要作用。在本研究中，我们将HGF基因传递到肝硬化大鼠肝脏，并研究HGF基因转导是否能预防肝切除术后致死性肝衰竭。方法：采用1%二甲基亚硝胺（DMN）诱导肝硬化，每周连续3天，连续4周。末次注射DMN 3 d后，用日本血凝病毒（HVJ）脂质体经门静脉左夹持将含CMV启动子的HGF基因20 μg导入肝脏。对照大鼠按相同方法注射**S。三分之二的肝切除…根据Higgins和Anderson描述的程序，在HGF基因或PBS注射后3天形成。于三分之二肝切除术后6、12、24、72、168小时处死大鼠，取血、取肝组织。应用增殖细胞核抗原（PCNA）免疫组化方法研究肝细胞DNA合成。采用TUNEL法检测肝细胞凋亡。Western blotting检测促凋亡和抗凋亡蛋白Bax、Bcl-xL的表达。结果：对照组大鼠在肝切除后6 h开始死亡，3 d死亡的大鼠占31.5%。另一方面，HGF基因转导显著提高了死亡率，92.7%的大鼠在7天存活。肝切除术后3 d， PCNA检测HGF基因转染大鼠肝脏中肝细胞的DNA合成高于对照大鼠肝脏，但各组间残肝体积差异无统计学意义。TUNEL法在对照组肝切除术后早期观察到大量凋亡的肝细胞，而在HGF基因转移的肝脏中观察到少量凋亡的细胞。虽然抗凋亡蛋白Bcl-xL在肝切除术后的早期正常肝脏中表达显著增加，但在本研究中，这些蛋白在肝硬化大鼠肝脏中被显著抑制。肝切除术后将HGF基因转入肝硬化可显著改善这些蛋白的表达。另一方面，促凋亡蛋白Bax在正常大鼠肝切除术后3天未见表达，但在对照组和HGF转导的大鼠肝切除术后，该蛋白已表达。HGF基因转导对肝硬化大鼠Bax蛋白无明显影响。结论：肝硬化部分切除肝后，HGF基因治疗可有效阻止肝细胞凋亡，提示HGF是一种抗凋亡剂而非促进再生因子。HGF的这种作用可能抑制了部分肝切除术后早期病程的死亡率。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