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Laboratory Animal Science of Gene Therapy Using Mutant Mice for T-cell Apoptosis

使用突变小鼠进行 T 细胞凋亡基因治疗的实验动物科学

基本信息

批准号：
07458225
负责人：
WATANABE Tomomasa
金额：
$ 4.67万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07458225/
关键词：
mouse Fas antigen lpr gene T-cell gene therapy apoptosis Western blot Northern blot ウエスタンブロットノーザンブロット精巣遺伝子的多型 1pr遺伝子

项目摘要

Vectors from retroviruses are known to transfer gene effectively into cells. However, the therapeutic use of viruses entails a problem as gene delivery vehicles, because retroviruses might disrupt DNA of the host cells when they infect, and would cause potentially harmful results. On the other hand , nonviral methods of gene transfer would be suitable for repeated use, since they do not result in the immune responses. Liposome is one of nonviral vector.It is considered that a gene therapy using Fas antigen in vivo is an important way to eleminate or regress tumors through apoptosis. A mouse lymphoproliferation (lpr) , which is a nonfunctional mutation of Fas antigen, has been assigned to chromosome 19. Transcript of the Fas antigen in MRL-lpr mice is nearly expressed in the thymus and liver compared to that in normal mice because an insertion by an early transposable elment is occured. The clinical syndrome of MRL-lpr mice is characterized as lymphoadenopathy, hypergammaglobulinaemia a … More nd arthritis, which resembles human systemic lupus erythematosus. In this study the expression vectors carrying FAS antigen cDNA are constructed in the correct orientation named pEF-Fas and pCMV-Fas. First, it became apparent in vitro experiment and Western blot analysis that Fas antigen protein is expressed in COS-l cells transfected by lipofectin and pEF-Fas or pCMV-Fas complexes. Then MRL-lpr mice were used for investigating the effect of Fas antigen expression by means of transfection of the expression vectors enclosed in cationic liposomes. After injected through tail vein, the vectors carrying Fas antigen cDNA-liposome complex were successfully transferred in mainly lung, liver, spleen, kidney and pancreas as detected by Southern blot analysis. Furthermore, the RNA transcripts were found in lung and liver by Norhtern blot analysis, but not observed in any other organ. Fas antigen protein, however, was not observed even in lung and liver by Western blot analysis. Finally 66% partial hepatectomy was attempted to find Fas antigen protein effectively, but could not detected. Future study is nessesary to investigate the reason why transferred DNA-liposome complex was not expressed as protein, and to search the way to express Fas antigen in vivo. Less

已知逆转录病毒载体可以有效地将基因转移到细胞中。然而，病毒的治疗用途作为基因传递载体存在一个问题，因为逆转录病毒在感染时可能会破坏宿主细胞的DNA，并会导致潜在的有害结果。另一方面，基因转移的非病毒方法适合重复使用，因为它们不会导致免疫反应。脂质体是非病毒载体之一。体内Fas抗原的基因治疗被认为是通过细胞凋亡消除或消退肿瘤的重要途径。小鼠淋巴增殖（lpr）是 Fas 抗原的非功能性突变，已被分配到 19 号染色体。与正常小鼠相比，MRL-lpr 小鼠中 Fas 抗原的转录物几乎在胸腺和肝脏中表达，因为发生了早期转座元件的插入。 MRL-lpr 小鼠的临床综合征的特征是淋巴结病、高丙种球蛋白血症和关节炎，类似于人类系统性红斑狼疮。在本研究中，以正确的方向构建携带FAS抗原cDNA的表达载体，命名为pEF-Fas和pCMV-Fas。首先，在体外实验和蛋白质印迹分析中发现，Fas抗原蛋白在由lipofectin和pEF-Fas或pCMV-Fas复合物转染的COS-1细胞中表达。然后用MRL-lpr小鼠通过阳离子脂质体包封的表达载体转染来研究Fas抗原表达的效果。通过尾静脉注射后，经Southern印迹分析检测，携带Fas抗原cDNA-脂质体复合物的载体主要成功转移到肺、肝、脾、肾和胰腺中。此外，通过 Norhtern 印迹分析在肺和肝脏中发现了 RNA 转录物，但在任何其他器官中均未观察到。然而，通过蛋白质印迹分析，即使在肺和肝中也没有观察到Fas抗原蛋白。最后尝试66%部分肝切除术有效寻找Fas抗原蛋白，但未能检测到。未来的研究有必要探讨转移的DNA-脂质体复合物不表达为蛋白质的原因，并寻找在体内表达Fas抗原的方法。较少的