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[目的]构建CCNY和CCNX基因的真核表达载体并观察其在人肺癌细胞株A549中的表达及定位,为进一步探讨Cyclin Y、Cyclin X在肺癌中的细胞定位和功能奠定了基础。[方法]以人肺腺癌细胞株H1299cDNA为模板扩增CCNY和CCNX基因,并构建CCNY和CCNX过表达真核表达载体。应用荧光显微照相及Western Blot方法鉴定该细胞株中Cyclin Y、Cyclin X的定位及表达。[结果]成功构建pEGFP-N1/CCNY和pEGFP-N1/CCNX真核表达载体。荧光显微照相显示绿色荧光,Western Blot检测证实转染重组质粒细胞表达Cyclin Y、Cyclin X蛋白,Cyclin Y和Cyclin X分别定位于胞膜与胞核。A549-pEGFPN1细胞活性为1.31±0.03,而转染后的A549-CCNY pEGFPN1细胞活性为1.36±0.02,A549-CCNX pEGFPN1细胞活性为1.45±0.05(P<0.01)。[结论]Cyclin Y和Cyclin X在A549细胞株中定位具有明显的差异,而且胞浆定位的Cyclin X比细胞膜定位的Cyclin Y更能够促进A549细胞增殖。
[Objective] To construct eukaryotic expression vectors of CCNY and CCNX genes and observe their expression and localization in human lung cancer cell line A549, which laid the foundation for further study of the cellular localization and function of Cyclin Y and Cyclin X in lung cancer. [Method] The CCNY and CCNX genes were amplified from human lung adenocarcinoma cell line H1299 cDNA and the eukaryotic expression vectors of CCNY and CCNX were constructed. The localization and expression of Cyclin Y and Cyclin X in this cell line were identified by fluorescence microscopy and Western Blot. [Results] The eukaryotic expression vectors pEGFP-N1 / CCNY and pEGFP-N1 / CCNX were successfully constructed. Fluorescence micrographs showed green fluorescence. Western Blot showed that the expression of Cyclin Y, Cyclin X protein, Cyclin Y and Cyclin X were localized in the membrane and nucleus respectively. The cell viability of A549-pEGFPN1 was 1.31 ± 0.03, while the activity of transfected A549-CCNY pEGFPN1 was 1.36 ± 0.02 and the activity of A549-CCNX pEGFPN1 was 1.45 ± 0.05 (P <0.01). [Conclusion] The expression of Cyclin Y and Cyclin X in A549 cell line has obvious difference, and the cytoplasmic localization of Cyclin X can promote the proliferation of A549 cell.