CyclinY和CyclinX在肺癌细胞株A549中的细胞定位  

Cell Localization of Cyclin Y and Cyclin X in Lung Cancer Cell Line A549

作　　者：江姝[1] 赵晓婷[1] 杨学惠[1] 顾勐 王玥[1] 岳文涛[1] 

机构地区：[1]北京市结核病胸部肿瘤研究所细胞生物学研究室,北京101149

出　　处：《结核病与胸部肿瘤》2012年第1期6-10,共5页Tuberculosis and Thoracic Tumor

摘　　要：背景与目的CyclinY及其同源异构体CyclinX是新发现的细胞周期蛋白，与肿瘤细胞增殖关系密切。但其细胞定位及与肿瘤增殖的关系尚不清楚。为明确CyclinY、CyclinX在肺癌中的细胞定位和作用机理，我们构建了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。方法以人肺腺癌细胞株H1299cDNA为模板，扩增CCNY和CCNX基因；PCR产物与pGEM-T载体连接，经测序验证后将插入片段亚克隆到真核表达载体pEGFP-N1。脂质体转染A549细胞株，应用荧光显微照相及WesternBlot方法鉴定该细胞株中CyclinY、CyclinX的定位及表达。结果pECFP-NI／CCNY和pEGFP-N1／CCNX重组子经酶切及DNA测序鉴定，目的基因CCNY和CCNX的序列正确，真核表达载体成功构建。转染重组子的细胞株在荧光显微镜下观察均可见绿色荧光蛋白的表达。Westernblot检测证实转染重组质粒组有CyclinY、CyclinX蛋白表达，而转染空质粒组没有检测到两蛋白的表达。CyclinY主要定位于A549细胞的胞膜，CyclinX主要在胞核分布。结论成功构建真核表达载体pEGFP-NI／CCNY和pEGFP-NI／CCNX并能在人肺癌细胞株A549中表达，证实CyclinY和CyclinX在A549细胞株中定位具有明显的差异，为进一步研究CyclinY和CyclinX的细胞定位及作用机理奠定了基础。Bakground and Objective Cyclin Y and its homologous isomer Cyclin X are one of new cell cycle proteins that have been discovered in recent years, and they are closely related with tumor cell proliferation. However, the relationship between their cellular localization and tumor cell proliferation is unclear. In order to determine the cellular localization and relative mechanisms of CCNY and CCNX in lung cancer, we constructed the recombinant eukaryotic expression vector pEGFP-N1/CCNY and pEGFP-N1/CCNX and explored their expression in human lung cancer cell A549. Methods CCNY and CCNX genes were amplified by PCR using H1299 eDNA as a template; The aim gene fragments CCNY and CCNX from pGEM-T/CCNY and pGEM-T/CCNX were subcloned in enkaryotic expression vector pEGFP-N1, respectively. The recombinant plasmids pEGFP-N1/CCNY and pEGFP-N1/CCNX were identificated by double digestion and sequence analysis, and then they were transfected into A549 cell by LipofectamineTM 2000. The cellular localization and expression of CyclinY and Cyclin X were detected by fluorescence microscope and Western Blot. Results The construction of the recombinant plasimid pEGFP-NI/CCNY and pEGFP-NI/CCNX were proved successfully by restriction enzyme digestion analysis and DNA sequencing. After transfected by LipofectamineTM 2000, green fluorescent proteins pEGFP-NI/CCNY and pEGFP-N1/ CCNX were detected. Western blot analysis confirmed that Cyclin Y and Cyclin X were successfully expressed in A549 cell. In addition, the present study showed that ectopic expressed CCNY was enriched at the plasma membrane. In contrast, the CCNX was enriched in cytoplasm and nuclear. Conclusion The eukaryotic expression plasmid pEGFP-NI/CCNY and pEGFP-N1/CCNX have been successfully constructed and expressed in the A549 cell line. And we demonstrated ectopic expressed CCNY and CCNX are differently distributed in A549 cell. The present study will help further studies on the cancer relative mechanisms and the relationship between CCNY and CCNX.

关 键 词：CyclinY CyclinX 肺肿瘤:亚细胞定位 

分 类 号：R734.202[医药卫生—肿瘤]