3Δ40p53对p53促肿瘤细胞凋亡作用的影响
目的探讨p53亚体Δ40p53对p53促肿瘤细胞凋亡作用的影响。方法体外培养HCT116-p53-/-结肠癌细胞系（内源性表达Δ40p53）、HCT116-p53＋/＋结肠癌细胞系（内源性表达野生型p53）和H1299肺癌细胞系（p53缺失），感染p53腺病毒使p53过表达，转染Δ40p53质粒使Δ40p53过表达。逆转录聚合酶链反应（RT-PCR）检测Δ40p53和p53 mRNA，实时荧光定量PCR检测Δ40p53对p53转录水平的影响，Western blot检测相关蛋白的表达，免疫共沉淀检测Δ40p53与p53相互作用关系，Calcein-AM/PI染色和流式细胞术检测细胞凋亡水平。结...
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| Published in | 中华肿瘤杂志 Vol. 39; no. 5; pp. 332 - 338 |
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| Main Author | |
| Format | Journal Article |
| Language | Chinese |
| Published |
2017
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| Subjects | |
| Online Access | Get full text |
| ISSN | 0253-3766 |
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| Summary: | 目的探讨p53亚体Δ40p53对p53促肿瘤细胞凋亡作用的影响。方法体外培养HCT116-p53-/-结肠癌细胞系（内源性表达Δ40p53）、HCT116-p53＋/＋结肠癌细胞系（内源性表达野生型p53）和H1299肺癌细胞系（p53缺失），感染p53腺病毒使p53过表达，转染Δ40p53质粒使Δ40p53过表达。逆转录聚合酶链反应（RT-PCR）检测Δ40p53和p53 mRNA，实时荧光定量PCR检测Δ40p53对p53转录水平的影响，Western blot检测相关蛋白的表达，免疫共沉淀检测Δ40p53与p53相互作用关系，Calcein-AM/PI染色和流式细胞术检测细胞凋亡水平。结果Δ40p53在HCT116-p53-/-细胞中能够稳定转录并表达。实时荧光定量PCR和Western blot检测显示，Δ40p53对p53的转录及表达水平无影响。免疫共沉淀检测显示，Δ40p53与p53能相互结合。Calcein-AM/PI染色显示，H1299-Control组、HCT116-p53-/--Control组、H1299＋p53组、HCT116-p53-/-＋p53组、H1299＋奥沙利铂（Oxa）组、HCT116-p53-/-＋Oxa组、H1299＋p53＋Oxa组和HCT116-p53-/-＋p53＋Oxa组的细胞凋亡率分别为（2.50±0.47）%、（2.40±0.32）%、（5.20±0.58）%、（4.10±0.18）%、（22.40±1.73）%、（19.30±1.11）%、（29.90±1.15）%和（39.30±2.26）%。H1299＋p53＋Oxa组与HCT116-p53-/-＋p53＋Oxa组的细胞凋亡率差异有统计学意义（t＝3.721，P＝0.021）。Calcein-AM/PI染色显示，H1299-Control组、H1299＋Δ40p53组、H1299＋p53组、H1299＋p53＋ Δ40p53组、H1299＋Oxa组、H1299＋Δ40p53＋Oxa组、H1299＋p53＋Oxa组和H1299＋p53＋Δ40p53＋Oxa组的细胞凋亡率分别为（2.60±0.35）%、（2.20±0.17）%、（4.80±0.49）%、（4.90±1.10）%、（20.30±1.10）%、（19.60±1.45）%、（27.90±1.39）%和（35.20±1.43）%，H1299＋p53＋O |
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| Bibliography: | ObjectiveTo investigate the effect of Δ40p53, an alternative spliced isoform of p53 lacking the N-ter minus, on the pro-apoptotic function of p53.MethodsThe wild-type p53 was ectopically expressed in HCT116-p53-/- （endogenous Δ40p53 expression）, HCT116-p53＋ /＋ （wild-type p53） and H1299 （p53-null） cells by adenoviral delivery, while Δ40p53 plasmid were transfected into these cells to overexpress Δ40p53. The levels of Δ40p53 and p53 mRNA were detected by reverse transcription-polymerase chain reaction （RT-PCR） and quantitative PCR. The expression of related proteins was deter mined by Western blotting. The interaction of p53 and Δ40p53 was observed by co-immunoprecipitation assay. Calcein-AM/propidium iodide （PI） staining and flow cytometry were used to detect the apoptotic rate of tested cells in each group.ResultsHCT116-p53-/- cells expressed endogenous Δ40p53 isoform. Neither transcription nor protein expression of wild-type p53 was interfered by the increased expression of Δ40p53. Full length p53 and Δ40p53 |
| ISSN: | 0253-3766 |