Analysis of stress defense mechanism against oral cancer by using A170 gene knockout mouse

在本研究中，我们将重点放在氧化应激蛋白A170上，该蛋白被认为在口腔和颌面病变中发挥重要作用，因为它与癌基因和成骨细胞分化有关。我们计划1)构建A170基因敲除小鼠，2)从临床和基础角度分析包括A170在内的氧化应激蛋白的应激反应。1)关于基因敲除小鼠的构建，我们进行了以下步骤。从BAC/129Sv小鼠基因组文库中分离出A170基因克隆，并对其基因进行了鉴定。映射后，我们设计了A170-null目标向量。将修饰后的基因与载体通过电穿孔法导入胚胎干细胞。将基因改变的细胞注射到来自C57/B6N的胚胎囊胚中，然后植入代孕母亲体内。嵌合体产生了高刺毛百分比的外壳，这表明接受了基因突变。然后将这些嵌合小鼠与CS7/B6小鼠交配。携带刺鼠皮的F1后代表明是种系传播。FCR证实了种系传播。最后完成了Flmouse和敲除小鼠（F2）的杂交构建。2)在构建敲除小鼠的同时，研究应激剂对A170的诱导作用。我们还研究了过氧化氧还蛋白I （Prx I = MSP23）和其他应激诱导蛋白的表达，作为A170敲除小鼠分析的基础。此外，我们还从临床样本中研究了口腔癌的表达水平。

In this study we focused on the oxidative stress protein A170 which was estimated to play a important role in oral and maxillofacial lesions because it was associated with the oncogene and osteoblast differentiation. We planed 1) to construct A170 gene knockout mouse and 2) to analyze the stress : response of the oxidative stress proteins including A170 from clinical and basic viewpoints.1) About knockout mouse construction we carried out the following procedures. We isolated the genomic clone and characterized the gene of A170 from BAC/129Sv mouse genome library. After mapping we designed the A170-null targeting vector. The modified gene with the vector was introduced into the ES cells by electroporation. Genetically altered cells were injected into embryonic blastocysts derived from C57/B6N and then implanted into a surrogate mother. Chimeras were generated with a high agouti percentage coat which indicated acceptance of the gene mutation. These chimeric mice were then mated with CS7/B6 mice. The F1 offspring containing the agouti coat indicated germline transmission. The germline transmission was confirmed by FCR. Finally we mated Flmouse and knock out mouse (F2) construction was completed.2) While knockout mouse construction we investigated the induction of A170 by stress agents using the rat. We also investigated Peroxiredoxin I (Prx I = MSP23) and the other stress inducible protein expression as a foundation for A170 knockout-mouse analysis. Moreover, we also investigated the expression level of oral cancer from clinical samples.