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：本研究旨在探讨Wnt信号通路在肝细胞癌和结直肠癌中的变化，探讨利用Axin基因2调控Wnt信号通路治疗结直肠癌的方法。结果(1)结直肠癌中β-catenin的免疫组化及β-catenin与Axin1的突变分析：结直肠癌中β-catenin的积累频率较高。此外，Axin1基因存在多个突变。(2)大鼠转移性肝癌模型的构建：由于先前建立的转移性肝癌模型不适用，我们采用裸鼠皮下肿瘤模型。(3) TCF/β-catenin依赖性Axin1表达载体（TOP-Axin）的构建。(4)将TOP-Axin导入培养细胞：用脂质体将TOP-Axin和PEF-BOS/myc-Axin转染至COS-7细胞。使用抗myc抗体和抗axin抗体，western blotting检测Myc-Axin的表达。而TOP-Axin的表达量不足。GSK3-β抑制剂诱导β-catenin积累，但未诱导TOP-Axin表达。我们决定在动物模型上研究PEF-BOS/myc-Axin对结肠癌的影响。(5) HVJ-E基因转染：将GFP表达载体导入培养细胞，导入效率约为10%-20%，但不能获得足够的效率。(6) HVJ-E将Axin基因导入动物模型：使用HVJ-E - 3将PEF-BOS/myc-Axin载体注入皮下肿瘤后，免疫组织化学无法用抗Axin和抗myc抗体证实Axin的表达。展望未来，我们将在体内构建axin -仙台病毒载体用于动物模型。

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.