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

使用 HVJ-E 引入 Axin 基因和 TCF 诱饵开发结直肠癌转移性肝癌的基因治疗

基本信息

批准号：
14370391
负责人：
OKAJIMA Masazumi
金额：
$ 8.58万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370391/
关键词：
colorectal cancer metastatic liver cancer gene therapy Wnt signaling pathway Axin TCF

项目摘要

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.

1.目的：探讨Wnt信号通路在肝细胞癌和结直肠癌中的改变，为Axin基因调控Wnt信号通路治疗结直肠癌奠定基础。2.结果（1）结直肠癌组织中β-catenin的免疫组化及β-catenin和Axin 1的突变分析：结直肠癌组织中β-catenin的表达明显增高。此外，Axin 1基因存在多处突变。（2）大鼠转移性肝癌模型的建立：由于以往建立的转移性肝癌模型不适用，我们采用裸鼠皮下移植瘤模型。（3）TCF/β-catenin依赖性Axin 1表达载体（TOP-Axin）的构建。（4）TOP-Axin的转染：脂质体介导TOP-Axin和PEF-BOS/myc-Axin转染COS-7细胞。使用抗myc和抗Axin抗体通过蛋白质印迹法清楚地检测Myc-Axin表达。然而，TOP-Axin表达未被充分检测到。虽然GSK 3-β抑制剂诱导β-catenin的积累，但不诱导TOP-Axin的表达。我们决定在动物模型中检查PEF-BOS/myc-Axin对结肠癌的作用。（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%，不能获得足够的效率。（6）用HVJ-E将Axin基因导入动物模型中：用HVJ-E将PEF-BOS/myc-Axin载体注射到皮下肿瘤中，用抗Axin和抗myc抗体检测，均未证实Axin的表达。