Genetic Analysis of Spontaneously Hepatitic and Cancerous Rat and Separation of the Unique Genes Into Other Lines as Ricombinant and Congenic Strains

摘要我们研究了一种新的突变大鼠（LEC）肝损伤，然后自发发展为肝细胞癌的肝谷胱甘肽依赖性酶的表达与异常铜积累的关系。我们发现，LEC大鼠肝脏中铜浓度逐渐增加到49倍，但随后下降约20倍，与年龄匹配的正常大鼠相比。LEC大鼠肝脏中还原型谷胱甘肽水平随年龄增长而增加。老年LEC大鼠谷胱甘肽S-转移酶活性不变，硒依赖的谷胱甘肽过氧化物酶活性下降。在LEC大鼠肝脏中，我们发现谷胱甘肽S-转移酶1-1的相对表达量降低了2 ~ 5倍，而谷胱甘肽S-转移酶2-2的相对表达量增加了5 ~ 8倍。采用蛋白质印迹法测定大鼠肝胞浆中谷胱甘肽S-转移酶亚基的水平。的r 关于我们 结果表明，在大鼠的整个生命过程中，Ya亚基的相对含量约为正常大鼠的一半，而Yc亚基的相对含量在1月龄时略低，但在4岁时，LEC中的Yc亚基的相对含量比正常大鼠高2倍。通过北方印迹也分别观察到编码Ya和Yc亚基的poly（A）RNA的相应减少和增加。Southern杂交分析显示Yc亚基没有基因扩增。我们的数据表明，谷胱甘肽S-转移酶2-2的过度表达是由于LEC突变大鼠中异常铜积累对YaYc基因家族的转录阶段的选择性调节。LEC大鼠似乎具有降低的血清IgG含量，尽管IgM和伊加水平正常，而IgG缺乏由命名为Igsr-1的单隐性基因控制（松本等，1989）。我们最近发现LEC大鼠胸腺中CD 4 ^+8^+细胞向CD 4 ^+8^-细胞的成熟受阻，导致CD 4 ^+ T细胞数量显著减少。可以推测，MHC II类抗原在胸腺中不表达，但该抗原在LEC胸腺中表达，LEC胸腺表现为髓质发育不全（Agui等，出版中）。然而，MHC II类分子的功能性突变可能会导致从CD 4 ^+8^+ T细胞到CD 4 ^+8^-T细胞的阳性选择缺陷。此外，研究辅助性T细胞缺乏和选择性IgG缺乏（Igsr-1）之间的关系是有意义的，因为低IgG水平可能是辅助性T细胞缺乏的结果。因此，在LEC大鼠可用作免疫缺陷研究的动物模型之前，必须对其新突变进行遗传表征。在本研究中，我们使用LEC和正常（WKAH或BN）大鼠的F_1和回交后代进行了这种辅助性T细胞免疫缺陷的遗传分析，以确定是否单个基因负责缺陷。并对该免疫缺陷基因与Igsr-1、RT-1等基因座进行了连锁分析，结果表明，肝炎和免疫缺陷基因的重组株和同源株可用于免疫缺陷与肝癌发生机制的研究。我们现在正在制作14 F代的CXA重组大鼠和4 N代的第三个同类品系。这些新菌株将在未来1年内建立，并且必须对上述研究非常有用。少

AbstractWe have examined for expression of the liver glutatione-dependent enzyme in relation to abnormal copper accumulation in a novel mutant rat (LEC) in which liver injury occurs and then hepatocellular carcinoma spontaneously develops. We have found that the copper concentration gradually increased up to 49-fold, but then decreased about to 20-fold in LEC rat liver as compared to that in age-matched normal rat. Levels of reduced glutathione were increased in LEC rat liver with age. The activity of glutathione S-transferases was unchanged, but that of selenium-dependent glutathione peroxidase decreased in LEC rat at the old age. We have found that relative expression of the glutatione S-transferase 1-1 was about 2 to 5-fold decrcase, while that of glutathione S-transferase 2-2 was 5 to 8-fold increase in the elution profiles from chromatofocusing in LEC rat liver. The levels of the subunits of glutathione S-transferases in rat liver cytosol were determined by Western blotting. The r … More esult shows that the relative content of the Ya subunit is about a half through the life time, while that of the Yc subunit is somewhat lower at the age of 1 month, but then 2-fold higher in LEC than in normal rat at the ages of 4. A corresponding decrease and increase in poly (A) RNAs coding for the Ya and Yc subunits were also observed by Northern blotting, respectively. Southern blot analysis showed no gene amplification for the Yc subunit. Our data suggest that the overexpression of glutathione S-transferase 2-2 is due to a selective regulation of the transcription stage for the YaYc gene family by abnormal copper accumulation in LEC mutant rat.LEC rats appear to have a decrease serum IgG content in spite of normal levels of IgM and IgA, and the IgG deficiency is controlled by a single recessive gene which is designated as Igsr-1 (Matsumoto et al. 1989). We recently found that LEC rats have a markedly decreased number of CD4^+ T-cells due to arrest of maturation from CD4^+8^+ cells to CD4^+8^- cells in the thymus. It might be supposed that MHC class II antigen is not expressed in the thymus, but the antigen was expressed in LEC thymus which showed hypoplasia of the medulla (Agui et al. In press). However, there was a possibility that a dysfunctional mutation in MHC class II might cause a defect in positive selection from CD4^+8^+ to CD4^+8^- T-cells. Furtheremore it is of interest to investigate a relationship between helper T-cell deficiency and the selective IgG deficiency (Igsr-1), because low IgG level could be a consequence of the helper T-cell deficiency. Therefore, before LEC rats can be used as an animal model for studies on immunodeficiency, their novel mutation must be genetically characterized. In this study, we carried out genetic analyses of this T helper immunodeficiency using F_1 and backcross progeny of LEC and normal (either WKAH or BN) rats to determine if a single gene is responsible for the deficiency. We also carried out linkage analyses on the gene for this immunodeficiency and several loci including Igsr-1 and RT1 loci.In conclusion, recombinant and congenic strains for hepatitis and immunodeficiency genes must be useful for studies on mechanisms of hepatocellular carcinoma in relation to immunodeficiency. We are now making CXA recombinant rats with 14F generation and third congenic strain with 4N generation. These new strains will be established during further 1 year and must be very useful for the studies described above. Less