稳定沉默CUL5基因的CNE2细胞系建立

The Construction of CNE2 Cell Line with CUL5 Stably Silenced

目的:建立稳定沉默CUL5基因的CNE2细胞系,探讨CUL5在连续分割照射下鼻咽癌细胞加速再增殖现象中所起的作用。方法:设计并合成能表达针对CUL5基因的shRNA的DNA序列,构建真核表达载体;脂质体转染重组质粒至人鼻咽癌细胞株CNE2;G418筛选阳性细胞并扩大培养;RT-PCR检测连续分割照射前后阳性细胞CUL5基因沉默效果。结果:经限制性内切酶酶切和DNA测序证实质粒中插入的为所需序列;G418筛选所得阳性细胞经RT-PCR证实照射前后均能抑制CUL5基因表达。结论:成功建立CUL5基因沉默的CNE2细胞系。

Objective： To construct CNE2 cell line with CUL5 gene stably silenced and confirm the role of CUL5 gene playing on NPC accelerated proliferation in continuous fractioned irradiation. Methods： To design and synthesize DNA sequences encoding shRNA . The vector expressing siRNA was constructed and transfected into nasopharyngeal carcinoma cell line CNE2 by liposome. Successfully transfected cells selected by G418 were cultured. RT-PCR was performed to verify the interfering effectiveness of CUL5 gene in the constructed cell line before and after irradiation. Results： DNA sequencing showed that the recombinant plasmids identified by restricted incision enzymes were successfully constructed. It was indicated that CUL5 gene expression was inhibited both before and after irradiation by the means of detecting CUL5 gene expression in CNE2 transfected with recombinant plasmid by RT-PCR and analyzing the result of electrophoresis strip through gel imaging system, and correcting by internal parameter β-actin. Conclusion ： CNE2 cell line with CUL5 gene silencing was successfully constructed.