p27kip1基因诱导食管癌细胞凋亡实验研究

Study of adenovirus mediated human p27kip1 antioncogene on the apoptosis in esophageal carcinoma cell lines

目的 探讨p2 7基因诱导人食管癌细胞凋亡的作用。方法 构建重组体腺病毒Ad p2 7kip1,转移到培养的人食管癌细胞EC 970 6 ,用流式细胞术、DNA片段分析法、TUNEL法观察Ad p2 7kip1诱导EC 970 6细胞凋亡的作用。 结果 成功构建重组体腺病毒Ad p2 7kip1,病毒滴度为 1.2 4× 10 12 CFU/ml,在感染倍数≥ 5 0感染强度时 ,即可达到 10 0 %的转导效率。Ad p2 7kip1转染食管癌细胞后 ,流式细胞术检测在G1期前出现亚倍体凋亡峰。细胞DNA抽提电泳后发现凋亡特征性梯度。TUNEL法检测凋亡指数分别为 37.3± 3.4 (Ad p2 7kip1组 )及 1.3± 0 .2 (空白对照组 ) ,差异有显著性(P <0 .0 1)。结论 p2 7可有效诱导食管癌细胞凋亡 。

Objective To study the effects of p27kip1 gene on the apoptosis in esophageal carcinoma cell lines EC 9706. Methods A replication deficient adenovirus vector encoding p27kip1(ad p27kip1) was constructed and human esophageal carcinoma cell lines EC 9706 were transduced in vitro. The cell apoptosis were determined by flow cytometry, TUNEL technique and DNA fragmentation analysis. Results Ad p27kip1 containing the p27kip1 sequence was successfully constructed and the virus titer was 1.24×10 12 CFU/ml. The transduction efficiency was 100% when multplicity of infection≥50. FCM analysis revealed a sub G 1 cell peak in ad p27kip1 transduced esophageal carcinoma cell lines. Agarose electrophoresis showed marked ladder. The difference of apoptotic index between the ad p27kip1 group and the control group was statistically significant(37.3±3.4 vs. 1.3±0.2, P <0.01). Conclusions p27kip1 gene could induce the apoptosis in esophageal carcinoma cell lines EC 9706, This study provides the basis for the future esophageal carcinoma treatment based on p27kip1 cDNA strategy.