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目的检测Canstatin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Canstatin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Can-statin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,Canstatin在鼻咽癌组织和细胞中表达下调或缺失。RT-PCR法成功获得Canstatin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Gen-Bank中Canstatin基因相应序列比较,100%同源。结论Canstatin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Canstatin基因,为Canstatin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 changes of Canstatin gene during the pathogenesis of nasopharyngeal carcinoma and clone its coding sequence. Methods Semi-quantitative RT-PCR was used to detect the expression of Canstatin in nasopharyngeal carcinoma, nasopharyngeal carcinoma cells and normal nasopharyngeal tissues, and the differences between them were compared. The PCR primers containing restriction sites were designed. The coding sequence of Can-statin protein 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, Canstatin expression was down-regulated or down-regulated in NPC tissues and cells. The full-length cDNA sequence of Canstatin gene coding region was successfully obtained by RT-PCR. The recombinant cloned plasmid was 100% homologous to the corresponding sequence of Canstatin gene in Gen-Bank after DNA sequencing. Conclusion Canstatin is down-regulated or down-regulated in NPC tissues and cells. Successful cloning of Canstatin gene in nasopharyngeal tissue by T / A technology and laying a foundation for Canstatin subcloning into eukaryotic expression vector pEGFP-N1 to explore its role in the growth and metastasis of nasopharyngeal carcinoma.