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目的:检测Tumstatin基因在鼻咽癌发病过程中的表达变化及克隆其编码序列。方法:利用半定量RT-PCR方法检测Tumstatin在鼻咽癌组织、鼻咽癌细胞和正常鼻咽组织中的表达,比较它们之间的表达差异。设计含酶切位点的PCR引物,利用RT-PCR方法从相对正常鼻咽组织中获取Tumstatin蛋白编码序列,T/A克隆入pMD18载体中。利用PCR和酶切鉴定获得阳性重组子。重组子最后经测序证实。结果:与相对正常鼻咽组织相比,Tumstatin在鼻咽癌组织和细胞中表达下调或缺失。RT-PCR法成功获得Tumstatin基因编码区全长cDNA序列。重组克隆质粒插入片段经DNA测序后与GenBank中Tumstatin基因相应序列比较,100%同源。结论:Tumstatin在鼻咽癌组织和细胞中表达下调或缺失。采用T/A技术成功克隆鼻咽组织中Tumstatin基因,为下一步将Tumstatin亚克隆入真核表达载体pEGFP-N1,探索其在鼻咽癌生长和转移中的作用奠定了基础。
Objective: To detect the expression of Tumstatin gene during the pathogenesis of nasopharyngeal carcinoma and clone its coding sequence. Methods: The expression of Tumstatin in nasopharyngeal carcinoma, nasopharyngeal carcinoma cells and normal nasopharyngeal tissues was detected by semi-quantitative RT-PCR. The difference of expression between them was compared. PCR primers with restriction sites were designed. Tumstatin protein coding sequence was obtained from normal nasopharyngeal tissues by RT-PCR. The T / A cDNA was cloned into pMD18 vector. Positive recombinant was obtained by PCR and restriction enzyme digestion. Recombinants were finally confirmed by sequencing. Results: Compared with normal nasopharyngeal tissues, Tumstatin expression was down-regulated or down-regulated in NPC tissues and cells. RT-PCR method successfully obtained Tumstatin gene coding region full-length cDNA sequence. The recombinant cloned plasmid was 100% homologous with the corresponding sequence of Tumstatin gene in GenBank after DNA sequencing. Conclusion: Tumstatin is down-regulated or down-regulated in NPC tissues and cells. The successful cloning of Tumstatin gene in nasopharyngeal tissue by T / A technique was the foundation for the next step of subcloning Tumstatin into eukaryotic expression vector pEGFP-N1 and exploring its role in the growth and metastasis of nasopharyngeal carcinoma.