Wider application of small-for-size partial liver grafting by gene transfer using teromerase reverse transcriptase (TERT).

使用端粒酶逆转录酶（TERT）进行基因转移的小型部分肝移植的更广泛应用。

• 批准号: 13671243
• 负责人: SOEJIMA Yuji
• 金额: $ 2.3万
• 依托单位: Grant-in-Aid for Scientific Research (C)(2)
• 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-13671243/
• 关键词: TERT; Ex vivo gene trancefer; 30% partial liver transplantation; small-for-size injury; テロメラーゼ; 遺伝子導入; 生体肝移植; 過小グラフト; 肝移植; electroporation; Splenopexy

1. Development of rat liver transplantation model using small-for-size graft:Rat liver transplantation model using small-for-size graft (30% of whole liver volume) was established, in which 100% 3-day mortality was assured.2. Development of gene-transfer technique:(1) Gene transfer using electroporation (EP) technique:An EP-mediated teromerase reverse transcriptase (TERT) gene transfer into human hepatocyte primary cultrure was carried out. A 10% of transfection effectivcess was achieved. However, gene transfer of TERT did not extend the survival of the cells. A direct damege to hepatocytes by EP technique was suggested.(2) Gene transfer into cold-preserved rat liver:Ex vivo EP-mediated or hydrodynamic gene transfer into a cold-preserved (UW solution, 4℃) rat liver graft was evaluated using rat liver transplantation model. The luciferase gene expression in the graft after liver transplantation was found to be increased. However, significant graft damage due to a gene transfer was observed.Therefore, development of an alternative strategy using nano-carrier was indicated.(3) In vivo IL-12 gene transfer using EP technique under immunosuppression:Using mice model carrying hepatoma cells (MH134), in vivo IL-12 gene transfer using EP technique was carried out under immunosuppression by FK506. Intratumoral administration of mIL-12 vector elevated intratumoral IL-12 and IFN-γ on the days 7 and 14 of transfer and significantly inhibited not only the growth of HCC, but also the angiogenesis in the tumor through expression of antiangiogenetic chemokaine IP-10.(4) Gene transfer using hydrodynamic method:As an alternative to EP technique, gene transfer using hydrodynamic method was established. Expression of inciferase gene peaked at 4 hour after injection. The mechanism of the method has not been elucidated so far, thus further investigation was indicated.

1.大鼠小体积肝移植模型的建立：建立大鼠小体积肝移植模型（30%全肝体积），3天死亡率100%.基因转移技术的研究进展：（1）电穿孔（EP）基因转移技术：采用EP介导的方法将端粒酶逆转录酶（TERT）基因导入原代培养的人肝细胞。转染效率达10%。然而，TERT的基因转移并没有延长细胞的存活。提示EP技术可直接损伤肝细胞。(2)冷保存大鼠肝脏中的基因转移：使用大鼠肝移植模型评估离体EP介导或流体动力学基因转移到冷保存（UW溶液，4℃）大鼠肝移植物中。肝移植后移植物中荧光素酶基因表达增加。然而，由于基因转移，观察到显着的移植物损伤。因此，使用纳米载体的替代策略的发展被指示。(3)免疫抑制条件下EP法体内IL-12基因转移：采用荷肝癌小鼠模型（MH 134），在FK 506免疫抑制条件下进行EP法体内IL-12基因转移。肿瘤内给予mIL-12载体在转移的第7天和第14天升高了肿瘤内IL-12和IFN-γ，并且不仅显著抑制HCC的生长，而且通过表达抗血管生成趋化因子IP-10抑制肿瘤中的血管生成。(4)使用流体动力学方法的基因转移：作为EP技术的替代，建立了使用流体动力学方法的基因转移。inciferase基因表达在注射后4小时达到高峰。该方法的机理至今尚未阐明，因此需要进一步研究。

项目成果

Tanaka S. et al.: "Oncogenic signal transduction and therapeutic strategy for hepatocellular carcinama."Surgery. 131. 142-147 (2002)

Tanaka S. 等人：“肝细胞癌的致癌信号转导和治疗策略。”外科。

Shinji Tanaka et al.: "Clinicopathologic risk factors for recurrence after a curative hepatic resectionfor henatocellular carcinoma"Surgery. 131. 148-152 (2002)

Shinji Tanaka 等人：“肝细胞癌治愈性肝切除术后复发的临床病理学危险因素”手术。

Soejima Y. et al.: "Feasibility of duct-to-duct biliary reconstruction in left-lobe adult-living-donor liver transplantation."Transplantation. 785. 557-559 (2003)

Soejima Y. 等人：“左叶成人活体供体肝移植中管对管胆道重建的可行性。”移植。

Yuji Soejima et al.: "Safety and Risk of Using Pediatric Donor Livers in Adult Liver Transplantation"Liver Transplantation. 7. 41-47 (2001)

Yuji Soejima 等人：“成人肝脏移植中使用儿童供体肝脏的安全性和风险”肝脏移植。

Tanaka S.et al.: "Clinicopathologic risk factor for recurrence after a curative hepatic resection for hepatocellular carcinama"Surgery. 131. 148-152 (2002)

Tanaka S.等人：“肝细胞癌治愈性肝切除术后复发的临床病理学危险因素”手术。