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目的:建立用于临床前药物安全性致癌评价的人类原癌基因c-Ha-ras转基因小鼠模型。方法:通过PCR方法克隆人的原癌基因c-Ha-ras,全长6.5 kb,含有4个外显子,以及该基因本身的启动子、调控序列和poly A信号序列;并将其通过原核注射注入C57BL/6J小鼠受精卵雄原核。通过PCR,real-time RT PCR和反转录cDNA测序比对等手段鉴定c-Ha-ras基因的插入和表达,并结合病理切片分析自发肿瘤的发生。结果:人类原癌基因c-Ha-ras在原核注射后的基因整合率达到1.6%,共得到人类c-Ha-ras基因的插入的首建鼠6只,其中3只得以成活,并且人类c-Ha-ras基因能够稳定遗传,建成C57-ras转基因小鼠系;C57-ras转基因小鼠反转录cDNA测序比对与人源c-Ha-ras一致;3个转基因小鼠系c-Ha-ras基因的表达分别是野生型小鼠的70.53、59.07和99.68倍;病理切片分析,6只首建鼠中3只有自发肿瘤发生。结论:本研究成功建立了C57/B6J背景的人类c-Ha-ras转基因小鼠模型。该C57-ras转基因小鼠的制作和用途已申请专利(CN101532018A),这是我国首个以临床前药物致癌性实验为目的的c-Ha-ras转基因小鼠,也是在我国建立符合ICH规范的、拥有自主知识产权的临床前药物安全性致癌评价替代方法体系的基础。
OBJECTIVE: To establish a human proto-oncogene c-Ha-ras transgenic mouse model for preclinical drug safety carcinogenesis. Methods: The human proto-oncogene c-Ha-ras was cloned by PCR. It had a total length of 6.5 kb and contained four exons, as well as the promoter, regulatory sequence and poly A signal sequence of the gene. Injection of C57BL / 6J mouse fertilized egg into pronucleus. The insertion and the expression of c-Ha-ras gene were identified by PCR, real-time RT PCR and reverse transcription cDNA sequencing, and the occurrence of spontaneous tumor was analyzed in combination with pathological sections. Results: The gene integration rate of human proto-oncogene c-Ha-ras reached 1.6% after prokaryotic injection. Six of them were obtained, of which three were survived, and human c -Ha-ras gene can be stably inherited, and the C57-ras transgenic mouse line was constructed; reverse transcriptase cDNA sequencing comparison of C57-ras transgenic mice was consistent with that of human c-Ha-ras; 3 transgenic mice c-Ha The expression of -ras gene was 70.53, 59.07 and 99.68 folds of that of wild-type mice, respectively. Pathological analysis showed that 3 of 6 mice initially developed spontaneous tumors. Conclusion: This study successfully established a human C-Ha-ras transgenic C57 / B6J transgenic mouse model. The C57-ras transgenic mice have been patented (CN101532018A) for the production and use of the transgenic mice, which is the first c-Ha-ras transgenic mouse in China to be used for the purpose of preclinical drug carcinogenicity experiments. , With independent intellectual property rights preclinical drug safety carcinogenic evaluation alternative method of system foundation.