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

基本信息

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

项目摘要

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愈方法之一，然而，大多数患者同时存在肝硬化和肝储备功能受损，因此，肝硬化肝切除术后常常会导致致命的肝功能衰竭。 (HGF) 已知是肝细胞中有效的有丝分裂原，在肝再生中发挥重要作用。在本研究中，我们将 HGF 基因导入肝硬化大鼠肝脏，并研究 HGF 是否存在。基因转导可预防肝切除术后致命的肝功能衰竭。方法：通过每周连续3天施用1%二甲基亚硝胺(DMN)诱导肝硬化，最后一次注射DMN后三天，注射20μg带有CMV启动子的HGF基因。使用日本血凝病毒(HVJ)-脂质体通过门静脉用左glisson夹子将其引入肝脏，对照大鼠在肝脏中注射**S。根据 Higgins 和 Anderson 描述的程序，三分之二肝切除术是在 HGF 基因或 PBS 注射后 3 天，即三分之二肝切除术后的第 6,12,24,72,168 小时形成的。处死大鼠并收集血样和肝细胞中的DNA合成，用增殖细胞核抗原（PCNA）的免疫组织化学进行研究。采用TUNEL法检测肝细胞中促凋亡和抗凋亡蛋白的表达，如Bax和Bcl-xL，并通过Western blotting分析。结果：对照组大鼠在肝切除后6小时开始死亡。，31.5% 的大鼠在 3 天后死亡，而 HGF 基因转导显着降低了死亡率，92.7% 的大鼠在 7 天时存活。肝切除后3天，用PCNA测定HGF基因转移大鼠肝脏的肝细胞合成高于对照大鼠肝脏，但各组之间残余肝脏体积没有观察到明显的差异，并观察到大量凋亡的肝细胞。 TUNEL 法观察对照组肝切除后的早期情况，而在 HGF 基因转移的肝脏中检测到很少的细胞凋亡，尽管抗凋亡蛋白 Bcl-xL 的表达显着增加。在肝切除术后早期的正常肝脏中，这些蛋白在肝硬化大鼠肝脏中受到抑制，本研究中HGF基因转移到肝硬化肝脏中显着提高了肝切除后这些蛋白的表达。肝切除后3天，正常大鼠肝脏中未见Bax，但该蛋白在肝切除后已在对照大鼠中表达，并且HGF基因转导未见明显的对抗作用。结论：肝硬化大鼠部分切除后，HGF基因治疗有效阻止肝细胞凋亡，表明HGF作为抗凋亡剂而非再生加速因子发挥作用，HGF的这种作用可能抑制死亡率。部分肝切除术后的早期。