过表达SGK3肝癌细胞BEL-7402在去甾体激素血清中的抗凋亡研究

Study on Anti- Apoptosis of Hepatocellular Carcinoma Cell BEL-7402 Growing in Charcoal Stripped Fetal Bovine Serum

目的:构建过表达血清与糖皮质激素调节激酶3(SGK3)的质粒以及稳定表达SGK3的肝癌细胞系BEL-7402,研究其在去甾体激素胎牛血清(FBS)中的抗凋亡能力。方法:PCR扩增SGK3基因,将扩增产物连接到p CDH载体,构建出p CDH-SGK3的慢病毒载体质粒,将其同空白对照p CDH-NC分别与慢病毒包装载体共转入293T细胞,包装成慢病毒p CHD-SGK3和p CDH-NC;将构建的慢病毒感染肝癌细胞BEL-7402并用嘌呤霉素筛选,Western印迹检测SGK3的表达;观察细胞在FBS及去甾体激素FBS中的生长情况。结果:包装出p CDH-SGK3重组慢病毒,此慢病毒感染肝癌细胞系BEL-7402后获得表达;CCK8实验表明过表达SGK3可促进肝癌细胞的生长,BEL-7042细胞中有雄激素的表达,去甾体激素FBS中细胞生长受到抑制,过表达SGK3可增强肝癌细胞在去甾体激素血清中的抗凋亡能力。结论:在肝癌细胞BEL-7402中过表达SGK3可促进细胞生长,可增强细胞在去甾体激素FBS中的抗凋亡能力。

Objective： To construct the vector carrying serum and glucorticoid regulated kinase 3（SGK3） coding sequence and to build BEL-7402 cell line stably overexpressing SGK3 protein, to study the anti-apoptosis ability of this cell line growing in charcoal stripped fetal bovine serum（FBS）. Methods： SGK3 gene was amplified by PCR and then linked to vector pCDH to construct the lentivirus vector of pCDH-SGK3. Vector pCDH-SGK3 and its empty vector pCDH-NC were respectively co-transfeeted into cells 293T with packaging carriers to produce len- tivirus-SGK3 and lentivirus-NC. Cells BEL-7402 were infected with produced lentivirus and filtered with puromy- cin. The expression level of SGK3 was tested by Western blot. Cell was cultured in DMEM with 10% FBS and 10% charcoal striped FBS respectively. Results： The vector carrying SGK3 coding sequence was successfully con- structed and BEL-7402 cell line stably overexpressing SGK3 protein was constructed. SGK3 could enhance the growth of cell BEL-7402, androgen receptor and the ability of anti-apoptosis cultured in the DMEM with 10% charcoal striped FBS. Conclusion： Overexpression of SGK3 in hepatocellular carcinoma cell could enhance its growth and the ability of anti-apoptosis cultured in the DMEM with 10% charcoal striped FBS.