摘要
目的研究Smac基因在肿瘤坏死因子相关的凋亡诱导配体(TRAIL)诱导的神经母细胞瘤(NB)SK-N-SH细胞凋亡中的增强作用。方法1.利用反转录(RT)-PCR从人胚肾293细胞中扩增人Smac基因全长cDNA,通过TA克隆构建重组T载体pGEM-T/Smac。2.将目的片段从重组T载体上酶切下来,构建真核细胞表达载体pcDNA3.1/Smac,并转染人NB SK-N-SH细胞。用新霉素G418筛选稳定细胞株SK-N-SH/Smac,并用Western blot鉴定Smac基因的过表达。3.用1 000μg/L的TRAIL作用于过表达Smac基因的SK-N-SH/Smac和普通的SK-N-SH细胞24 h。采用流式细胞分析检测细胞凋亡百分率。另设无TRAIL干预的SK-N-SH/Smac及正常SK-N-SH对照组。4.比色法测定各组细胞中caspase-8酶活性。结果1.成功建立稳定过表达Smac基因的NB克隆株SK-N-SH/Smac。2.流式细胞分析显示1 000μg/L的TRAIL作用24 h后,过表达Smac基因的SK-N-SH细胞凋亡百分率显著高于正常对照的SK-N-SH细胞[(44.8±4.71)%vs(23.6±3.05)%P<0.05]。3.比色法测定显示,同样在1000μg/L的TRAIL作用24 h后,过表达Smac基因的SK-N-SH/Smac细胞中,caspase-8的酶活性显著高于未转染的普通SK-N-SH细胞[(0.743±0.029)vs(0.476±0.031)P<0.05]。结论转染并过表达Smac基因可增强TRAIL对神经母细胞瘤SK-N-SH细胞的促凋亡作用。
Objective To determine the synergistic effect of Smac on augmenting apoptosis induced by tumor necrosis factor related apoptosis inducing ligand（TRAIL）in human neuroblastoma cell line SK-N-SH.Methods 1.The full length cDNA of human Smac gene was amplified by reverse transcription-polymerase chain reaction（RT-PCR）,PCR product was ligated with linearized vector pGEM-T-easy supplied in the TA cloning kit and sequenced.2.The correct cDNA of Smac was subcloned into eukaryocytic expression vector pcDNA3.1/myc-his and transfected into human neuroblastoma cell line SK-N-SH by lipofectamine-mediated transfection.After selected with G418,SK-N-SH/Smac cell clones which stably overexpressed Smac were isolated and characterized by Western blot.3.SK-N-SH and SK-N-SH/Smac were cultured in the medium containing 1 000 μg/L TRAIL and incubated for 24 hours.Flow cytometry was used to evaluate apoptosis.4.Caspase-8 relative activity was determined by colorimetric assay.Results 1.SK-N-SH/Smac cell clones which stably overexpressed Smac were successfully obtained.2.Apoptosis of SK-N-SH and SK-N-SH/Smac were induced by 1 000 μg/L TRAIL.The percentage of apoptosis of SK-N-SH/ Smac reached（44.8±4.71）% after the administration of 1 000 μg/L TRAIL for 24 hours and it was significantly higher than that of SK-N-SH which treated by the same dose of TRAIL for 24 hours [（23.6±3.05）%]（P〈0.05）.3.Compared with caspase-8 relative activity of SK-N-SH which were treated by 1 000 μg/L TRAIL for 24 hours,that of SK-N-SH/Smac was more enhanced [（0.743±0.029） vs（0.476±0.031） P〈0.05].Conclusion Transfection and overexpression of Smac gene could enhanced the pro-apoptosis effect of TRAIL on human neuroblastoma cell line SK-N-SH.
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
2008年第15期1173-1175,共3页
Journal of Applied Clinical Pediatrics
