Development of gene therapy for metastatic liver cancer from colorectal cancer by introduction of Axin gene and TCF decoy using HVJ-E

1.目的：研究Wnt信号通路在肝细胞癌和结直肠癌中的变化，探讨AXIN基因调控Wnt信号通路在结直肠癌治疗中的作用。结果(1)结直肠癌组织中β-连环蛋白的免疫组织化学及β-连环蛋白和Axin1的突变分析：β-连环蛋白在结直肠癌组织中有较高的积聚。此外，还发现了Axin1基因的多个突变。(2)大鼠转移性肝癌模型的建立：由于以前建立的转移模型不适用，所以我们采用裸鼠皮下移植瘤模型。(3)构建Tcf/β-连接素依赖的Axin1表达载体(TOP-Axin)。(4)将top-axin导入培养细胞：用脂质体将top-axin和PEF-BOS/myc-axin导入COS-7细胞。用抗myc和抗Axin抗体免疫印迹法检测到myc-axin的表达。然而，顶轴蛋白的表达没有被充分检测到。β抑制剂诱导β-连环蛋白积聚，但不诱导顶轴蛋白表达。我们决定在动物模型上检测PEF-BOS/myc-axin对结肠癌的影响。(5)将HVJ-E：GFP表达载体导入培养细胞，导入效率约为10%-20%，不能获得足够的效率。(6)通过HVJ-E将Axin基因导入动物模型：将pEF-BOS/myc-axin载体注射到皮下肿瘤后，用抗Axin和抗myc抗体进行免疫组织化学染色，不能证实Axin的表达。展望和现在的发展：我们将构建Axin-仙台病毒载体用于体内动物模型。

1.AIM :The aim of this study was to investigate the alteration in the Wnt signaling pathway in hepatocellular carcinoma and colorectal carcinoma and to develop of the treatment of colorectal cancer by regulation of Wnt signaling pathway using Axin gene.2.RESULTS(1)Immunohistochemisty of β-catenin and mutational analysis of β-catenin and Axin1 in colorectal cancer : High frequency of accumulation of β-catenin was observed in colorectal cancer. Furthermore, there were several mutations in Axin1 gene. (2)Construction of metastatic liver cancer model in rat : Because the metastatic model previously developed was not applicable, we used subcutaneous tumor model using nude mouse. (3)Construction of TCF/β-catenin dependent Axin1 expression vector (TOP-Axin). (4)Introduction of TOP-Axin to culture cells : TOP-Axin and PEF-BOS/myc-Axin were transfected to COS-7 cells by lipofectamine. Myc-Axin expression was clearly detected by western blotting using anti-myc and anti-Axin antibodies. However, TOP-Axin expression was not enough detected. Although GSK3-β inhibitor induced accumulation of β-catenin, TOP-Axin expression was not induced. We decided to examine the effect of PEF-BOS/myc-Axin on colon cancer in animal model. (5)Gene transfection using HVJ-E : GFP expression vector was introduced to the culture cells, introduction efficiency was approximately 10%-20%, and it couldn't obtained enough efficiency. (6)Introduction of Axin gene to animal model by HVJ-E : The expression of Axin couldn't be confirmed by immunohistochenistry using anti-Axin and anti-myc antibodies when PEF-BOS/myc-Axin vector was injected to subcutaneous tumor using HVJ-E.3.Future view and now on going :We are going to construct Axin-Sendai virus vector to use in animal model in vivo.