高表达JNK3促进表阿霉素诱导的细胞凋亡

High expression of JNK3 promote cell apoptosis induced by epirubicin

目的:构建人c-Jun氨基末端激酶3(JNK3)真核表达载体,转染HEK293细胞,建立稳定表达细胞系;以表阿霉素作为凋亡诱导剂研究JNK3与细胞凋亡关系.方法:以pD-BLeu-JNK3质粒为模板PCR扩增人JNK3基因全长,DNA重组技术将其定向插入到真核表达载体pcDNA3.1/myc-His B,经PCR和酶切鉴定,DNA测序正确,脂质体转染法转染HEK293细胞,通过G418选择培养,建立稳定表达细胞系.RT-PCR,Western Blot检测携带Myc标签的JNK3融合蛋白的表达,流式检测表阿霉素对稳定表达JNK3基因的HEK293细胞的促凋亡作用.结果:成功构建了pcDNA3.1-JNK3真核表达载体并已稳定转入HEK293细胞,建立了稳定表达细胞系,成功地表达目的基因;与对照细胞相比表阿霉素能明显促进高表达JNK3基因的HEK293细胞发生细胞凋亡.结论:JNK3稳定表达细胞系的建立和高表达JNK3促进表阿霉素诱导的细胞凋亡,为进一步研究JNK3的功能提供了良好的实验基础.

AIM： To construct the eukaryotic expression vector of the c-Jun N-terminal kinases 3 （JNK3）and stably transfect HEK293 cells with it, and to research the relationship between JNK3 and the cell apoptosis by using epirubicin induction. METHODS： The full-length JNK3 eDNA fragment was amplified by PCR from the pDBLeu-JNK3 plasmid and was inserted into eukaryotic expression vector pcDNA3. 1/myc-His B. After identification by PCR and restriction digestion, the recombinant plasmid was transfected into HEK293 cells by lipofectamine. After screening culture by C,418, a stably-transfected cell line was established, and the transcription and expression of the Myc tag JNK3- carrying fusion protein were identified by RT-PCR and Western Blot. The cell apoptosis induced by epirubicin was detected by flow cytometric analysis. RESULTS： The eukaryotic expression vector pcDNA3, 1-JNK3 was successfully constructed and the JNK3 gene was transfected stably into HEK293 cells. The JNK3 gene was expressed successfully. Epirubicin significantly promoted the apoptosis of JNK3 gene transfected HEK293 cells. CONCLUSION：The study provides a solid experimental foundation for further studies on the function of the JNK3 gene.