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目的利用α-crystallin启动子构建晶状体上皮细胞中JCV T抗原表达转基因鼠,试图建立晶状体自发肿瘤动物模型。方法利用限制性内切酶BclI分离pBSK(pBluescript SK)-T抗原,BamH I同尾连接插入以pBSK为载体的α-crystallin启动子下游,通过NciI酶切电泳分离带有α-crystallin启动子的T抗原核酸序列后显微注射入C57BL/6J小鼠受精卵中。PCR检测转基因阳性鼠及JCV病毒T抗原拷贝数,大体和组织学观察眼球及病变组织,免疫组织化学观察T抗原、p53和β-catenin表达。结果成功构建α-crystallin JCV T抗原表达质粒,转基因阳性鼠中2号鼠(αAT-2)拷贝数最高,该鼠眼球于11个月表现出肿瘤样改变,肿瘤细胞中T抗原、p53和β-catenin呈强阳性表达。结论 JCV T抗原直接诱发晶状体上皮肿瘤动物模型建立为JCV T抗原嵌入所致上皮肿瘤提供了直接实验依据,为JCV所致上皮肿瘤发生和演进分子机理研究奠定坚实基础,同时为抗肿瘤药物筛选及基因治疗监控提供了良好的动物模型。
OBJECTIVE: To construct JCV T antigen-expressing transgenic mice using α-crystallin promoter and to establish an animal model of spontaneous lens tumor. Methods pBSK (pBluescript SK) -T antigen was isolated by restriction endonuclease BclI. BamH I was ligated into the downstream of α-crystallin promoter with pBSK as a vector and cloned by NciI digestion. T antigen was microinjected into the fertilized egg of C57BL / 6J mice. PCR was used to detect the copies of T antigen of transgenic mice and JCV virus. The gross and histological changes of eyeball and pathological tissues were observed. The expressions of T antigen, p53 and β-catenin were observed by immunohistochemistry. Results The α-crystallin JCV T antigen expression plasmid was successfully constructed and the copy number of αAT-2 gene in transgenic mice was the highest. The mouse eyeball showed tumor-like changes at 11 months. T-antigen, p53 and β -catenin was strongly positive expression. Conclusion JCV T antigen directly induced lens epithelial tumor animal model established JCV T antigen imbedded epithelial tumor provides a direct experimental basis for JCV Epithelial neoplasia and the development of molecular mechanism of molecular basis for the study to lay a solid foundation for the screening of anti-tumor drugs and Gene therapy monitoring provides a good animal model.