趋化因子受体CXCR4 shRNA真核表达载体的构建

目的：构建趋化因子受体（CXCR4）特异的RNA干扰质粒载体。方法：根据GenBank数据库提供的CXCR4基因核苷酸序列,选择设计能转录短发夹状RNA（short hairpin RNAs,shRNA）的DNA序列,并与pWH1质粒载体连接,用酶切的方法进行鉴定;将构建成功的特异性表达载体（pWH1-CXCR4）稳定转染至人涎腺黏液表皮样癌Mc3细胞系,并应用RT-PCR和流式细胞术对细胞CXCR4的表达进行检测。结果：与Mc3细胞和转染空白质粒pWH1 Mc3细胞相比,转染pWH1-CXCR4表达载体的Mc3细胞CXCR4 mRNA和蛋白的表达明显降低。结论：构建的CXCR4特异性shRNA表达载体可有效的沉默CXCR4基因,为进一步研究CX-CR4表达对涎腺黏液表皮样癌增殖和转移的意义及治疗涎腺黏液表皮样癌奠定了基础。

The construction of eukaryotic expression vector of short hairpin RNA for CXCR4

Objective:To construct of eukaryotic expression vector of RNA interference specific for CXCR4. Methods: Genome sequences of CXCR4 gene were retrieved from Genbank and cDNA was designed coding expression of shRNA (small hairpin RNAs) for CXCR4 gene. The cDNA was synthesized and inserted into plasmid pWH1, and the recombinant pWH1-CXCR4 expression vector was identified by enzyme cutting method. Then, pWH1-CXCR4 expression vector were transfected into salivary mucoepidermoid carcinoma Mc3 cells. At last, the expression of CXCR4 in Mc3 transfected with pWH1-CXCR4 expression vector was evaluated by RT-PCR and flow cytometry analysis. Result:Compared with Mc3 cells and Mc3 cells transfected with plasmid pWH1, the Mc3 cells transfected with pWH1-CXCR4 expression vector showed a lower mRNA and protein expression of CXCR4. Conclusion:The constructed CXCR4 shRNA expression vector can block the expression of CXCR4 in salivary mucoepidermoid carcinoma cells, and it may be helpful for further study on the significance of CXCR4 on the proliferation, metastasis and experimental treatment of salivary mucoepidermoid carcinoma.