摘要
目的 研究p16 INK4A对A5 4 9肺小细胞腺癌细胞系表型的影响。方法 用pcDNA3 p16质粒转染 p16基因缺失的A5 4 9肺小细胞腺癌细胞系并通过Northernblot和Westernblot鉴定 p16基因的异位表达 ,通过流式细胞仪分析转染后细胞周期变化 ,利用Westernblot和SA β gal染色检测Rb蛋白磷酸化状态的改变和细胞表型变化。结果 证实了在A5 4 9中 p16 INK4A能稳定表达。p16 INK4A的表达能使细胞产生明显的G1期阻滞并伴随着Rb蛋白磷酸化状态的改变 ,Rb蛋白主要以低磷酸化形式存在。发生生长阻滞的转染细胞表现出一系列细胞衰老样表型 ,如细胞体积变大 ,细胞形态呈明显的扁平状态 ,并且存在衰老相关半乳糖苷酶阳性染色。结论 在A5 4 9细胞中表达 p16 INK4A能使细胞发生衰老样表型并提示 p16
Purpose: To investigate the effect of p16INK4A on the phenotype of A549 cell fine. Methods: A549 cell line was transfected with p16 INK4A and the stably transfected strain was identified with Northern blot and Southern blot. Cell cycle was analysied by flow cytometry. The phosphorylation state of retinoblastoma protein(Rb) and cell phenotype were determined by Western blot and SA-β-gal staining. Results: Stable transfection of p16INK4A was confirmed. Overexpression of p16 INK4A in A549 cell line caused a G1 phase arrest with the inhibition of phosphorylation of Rb protein. Significantly, the arrested cells displayed several phenotypic features that were considered as the characteristics of senescent cells. They had a large, flat appearance, and positive acidic β-galactosidase acivity. Conclusions: Our results suggested that the re-expression of p16INK4A in A549 cells caused an immediate cellular senescence-like phenotype.
出处
《复旦学报（医学版）》
EI
CAS
CSCD
北大核心
2004年第2期111-114,共4页
Fudan University Journal of Medical Sciences
