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

体内 HGF 基因转移促进肝硬化大鼠肝再生并抑制大规模肝切除术后的致死性肝衰竭

• 批准号: 13671358
• 负责人: UEKI Takahiro
• 金额: $ 2.24万
• 依托单位: Hyogo College of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671358/
• 关键词: Liver cirrhosis; Massive hepatectomy; Liver failure; HGF (hepatocyte growth factor); Gene therapy; Apoptosis; HGF

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

背景/目的：肝脏手术被认为是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脏储备功能受损。因此，大面积肝切除术常引起急性肝衰竭术后致死性肝衰竭。肝细胞生长因子（HGF）是肝细胞内一种强有力的促有丝分裂原，在肝再生中起重要作用。在本研究中，我们将HGF基因转染到肝硬化大鼠肝脏，并探讨HGF基因转导是否可以预防肝切除术后致死性肝功能衰竭。方法：用1%二甲基亚硝胺（DMN）诱导肝硬化，每周连续3天，共4周。在最后一次注射DMN后3天，使用日本血凝病毒（HVJ）-脂质体通过门静脉用左Glisson夹将20 μg具有CMV启动子的HGF基因导入肝脏。对照组大鼠按相同方法注射 **S。三分之二肝切除术 ...更多信息 根据Higgins和安德森描述的方法，在HGF基因或PBS注射后3天形成 ***。分别于2/3肝切除术后6、12、24、72、168 h处死大鼠，取血和肝组织。增殖细胞核抗原（PCNA）免疫组化法检测肝细胞DNA合成。采用TUNEL法检测肝细胞凋亡情况。结果：对照组大鼠肝切除后6 h开始死亡，3d死亡率为31.5%;另一方面，HGF基因转导显著改善了死亡率，7 d时大鼠存活率为92.7%。肝切除术后3d，肝细胞增殖细胞核抗原（PCNA）检测显示，HGF基因转染组大鼠肝细胞DNA合成量高于对照组，而各组大鼠的残肝体积无显著性差异。对照组肝切除术后早期TUNEL法检测到大量凋亡细胞，而转染HGF基因的肝切除术后早期仅检测到少量凋亡细胞。虽然抗凋亡蛋白Bcl-xL的表达已被证明在肝切除术后的早期阶段在正常肝脏中显著增加，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，HGF基因转移到异位肝脏中显著改善了这些蛋白的表达。另一方面，促凋亡蛋白Bax的表达在正常大鼠肝切除术后3天未观察到，但该蛋白在对照组和HGF转导组大鼠肝切除术后已经表达。结论：肝纤维化大鼠部分肝切除后，HGF基因治疗能有效地抑制肝细胞凋亡，提示HGF可能是一种抗凋亡剂而非再生促进因子。肝细胞生长因子的这种作用可能抑制了肝部分切除术后早期的死亡率。少

项目成果

植木孝浩: "疾病克服への細胞増殖因子研究:肝硬変治療とHGF"Molecular Medicine. 38. 320-327 (2001)

Takahiro Ueki：“细胞生长因子克服疾病的研究：肝硬化和 HGF 的治疗”《分子医学》38. 320-327 (2001)。

Takahiro Ueki: "Liver regeneration using Hepatocyte Growth factor"Geka. 63. 279-285 (2001)

Takahiro Ueki：“利用肝细胞生长因子进行肝脏再生”Geka。

Takahiro Ueki: "HGF Gene Therapy for Liver Cirrhosis"Jap.J Apheresis. 21. 122-126 (2002)

Takahiro Ueki：“肝硬化的 HGF 基因治疗”Jap.J Apheresis。

Takahiro Ueki: "Liver cirrhosis"Nippon Rinsho. 59. 152-156 (2001)

植木贵宏：《肝硬化》日本林正。

Takahiro Ueki: "Therapy for liver cirrhosis with HGF"Molecular Medicine. 88. 320-327 (2001)

Takahiro Ueki：“HGF 治疗肝硬化”分子医学。