血管形成抑制剂Canstatin在鼻咽癌中表达鉴定及基因克隆

Identification of the Expression of Antiangiogenic Factor Canstatin in NPC and Gene Clone

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 alteration of Canstatin gene in nasopharyngeal carcinoma （NPC） pathogenesis and clone the coding sequence （ CDS ） of Canstatin. Methods Semiquantitative RT - PCR was used to detect the expression differentiation of Canstatin among NPC tissues, NPC cells and normal nasopharyn- geal epithelium（NP）. PCR primers with the design of containing restriction endonuclease sites were utilized to amplify CDS sequence of Canstatin from human normal NP. The PCR product was then inserted into pMD18 vector. The positive recombinant was obtained through the identifcation of PCR amplification and restriction endonuclease Sacl digestion. The sequencc of recombinant was finally confirmed by sequence analysis. Results Compared with NP, decreased and absent expression of Canstatin was shown in NPC tissue and NPC cells. CDS sequence of Canstatin was successfully obtained by HT- PCR method. Inserted cDNA fragment confirmed by sequencing was identical with the Canstatin sequence of Genebank. Conclusion Downregulated and absent expression was displayed in NPC tissues and NPC cell. Canstatin gene was successfully cloned into pMD18 vector, which laid the basis for subcloning Canstatin into pEGFP - N1 vector and exploring the roles in the process of NPC growth and metastasis.