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

1.小体积大鼠肝移植模型的建立：建立了小体积(占全肝体积的30%)大鼠肝移植模型，3天内死亡率为100%。基因转移技术的发展：(1)电穿孔(EP)基因转移：将EP介导的反转录酶(TERT)基因转移到原代培养的人肝细胞中。获得了10%的转染率。然而，TERT的基因转移并没有延长细胞的存活时间。(2)冷保存大鼠肝脏的基因转移：采用大鼠肝移植模型，对冷保存(UW液，4℃)大鼠移植肝进行体外EP介导的基因转移或流体动力基因转移。肝移植后移植物中荧光素酶基因表达增强。(3)免疫抑制下用EP技术进行体内IL-12基因转移：利用FK506免疫抑制下的小鼠肝癌(MH134)模型，用EP技术进行体内IL-12基因转移。瘤内注射mIL-12载体后第7天和第14天肿瘤内IL-12和干扰素-γ水平升高，不仅显著抑制了肝癌的生长，而且通过表达抗血管生成趋化因子IP-10抑制了肿瘤的血管生成。(4)流体力学基因转移：作为EP技术的替代方法，建立了流体力学基因转移技术。Cinferase基因表达在注射后4h达到高峰。到目前为止，该方法的作用机理尚未阐明，因此需要进一步研究。

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.