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

已知来自逆转录病毒的载体将基因有效地转移到细胞中。然而，病毒作为基因递送载体的治疗用途带来了问题，因为逆转录病毒在感染时可能破坏宿主细胞的DNA，并可能导致潜在的有害结果。另一方面，基因转移的非病毒方法将适合重复使用，因为它们不会导致免疫应答。脂质体是一种非病毒载体，利用Fas抗原进行体内基因治疗被认为是通过凋亡消除或逆转肿瘤的重要途径。小鼠淋巴细胞增殖（lpr），这是一个无功能的突变Fas抗原，已被分配到19号染色体。与正常小鼠相比，MRL-lpr小鼠的Fas抗原转录本在胸腺和肝脏中几乎表达，这是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， ...更多信息 和关节炎，类似于人类系统性红斑狼疮。本研究以正确的方向构建了携带FAS抗原cDNA的表达载体pEF-Fas和pCMV-Fas。首先，体外实验和Western blot分析表明，Fas抗原蛋白在脂质体和pEF-Fas或pCMV-Fas复合物转染的COS-1细胞中表达。用阳离子脂质体包封的表达载体转染MRL-lpr小鼠，观察Fas抗原表达的影响。经尾静脉注射后，经Southern blot检测，Fas抗原cDNA-脂质体复合物载体主要在肺、肝、脾、肾和胰腺中成功转移。Norhtern印迹分析表明，在肺和肝中有RNA转录本，而在其他器官中没有观察到。然而，通过Western印迹分析，即使在肺和肝中也未观察到Fas抗原蛋白。最后66%的肝部分切除术后未能检测到Fas抗原蛋白。进一步的研究还需进一步探讨DNA-脂质体复合物不能表达Fas抗原的原因，以及寻找Fas抗原在体内表达的途径。少

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