摘要
目的 :研究p2 7kip1基因转移对食管癌的体内、外抑制作用 ,并探讨其抑癌机制。方法 :①构建携带人p2 7kip1cDNA的重组腺病毒 (Ad p2 7kip1) ;②将Ad p2 7kip1转染人食管癌细胞Eca970 6 ,MTT法、3 H TdR和3 H Leucine掺入试验检测对细胞增殖的影响 ,流式细胞仪 (FCM)、DNA片段分析检测对细胞周期和凋亡的影响 ,TRAPPCR -ELISA法检测端粒酶活性 ,免疫细胞化学法及Westernblotting法检测p2 7kip1和Survivin的表达 ;③Ad p2 7kip1直接注射入人食管癌裸鼠移植瘤中 ,观测抑瘤效应 ,并检测p2 7kip1和Survivin的表达。结果 :①Ad p2 7kip1转染食管癌细胞后 ,细胞增殖明显受抑制 ,G0 /G1期细胞比例增加 ,并出现明显的亚二倍体凋亡峰和特征性的凋亡梯状条带 ,细胞端粒酶活性降低 ,p2 7kip1表达增强 ,Survivin表达降低 ;②采用p2 7kip1基因治疗 ,食管癌裸鼠移植瘤生长明显受抑制 ,肿瘤生长抑制率达 6 4 .1% ,移植瘤中p2 7kip1表达增强 ,Survivin表达降低。结论 :p2 7kip1基因转移对食管癌具有较显著的体内、外抑制作用 ,其机制是增强p2 7kip1表达、抑制Survivin表达 ,使细胞产生G1期阻滞 ,抑制端粒酶活性 ,并诱导凋亡 ,表明该基因疗法是食管癌治疗的新途径。
Objective To investigate inhibitory ef fect of p27kip1 gene transfer on esophageal carcinoma in vivoa nd in vitroand its mechanisms. Methods ①The recombinant adenoviral vector with human p27kip1 cDNA(Ad-p27kip1) was con structed and prepared. ②Esophageal carcinoma cells Eca9706 were transfected by Ad-p27kip1. The effects of Ad-p27kip1 on cellproliferation were evaluated with MTT assay, 3H-TdR and 3H-Leucine incorporation. Cell cycle and ap optosis were detected by flow cytometry(FCM) and DNA fragmentation. Activity of telomerase was examined by TRAP PCR-ELISA. Expression of p27kip1 and Survivin we re detected by immunocytochemical technique and Western blotting.③The therapeut ic effects with intratumoral injection of Ad-p27kip1 into established tumors we re evaluated with growth curves and inhibitiory rates of tumor.Expression of p27 kip1 and Survivin were detected.Results ①After infected b y Ad-p27kip1,EC9706 cell proliferation was markedly inhibited. The percentage o f cells at G 0/G 1 phase was increased.Obviously apoptotic sub-G 1 peak and marked ladder were found. Activity of telomerase was obviously inhib ited. Expression of p27kip1 was increased and expression of Survivin was decreas ed. ②The growth of tumors in gene therapy group with p27kip1 were obviously sup pressed, the growth inhibition rate reached 64.1%.In addition,the expression of p27kip1 was increased and expression of Survivin was decreased in tumors after t ransfected with Ad-p27kip1. Conclusions p27kip1 gene the rapy mediated by adenovirus vector has significant inhibitory effect on esophage al carcinoma in vivoand vitro. The mechanisms include upregulation of p27kip1 expression, down regulation of Survivin expression, G 1 arrest, depression of telomerase activity and inducing apop tosis.The results indicate that p27kip1 gene therapy may be a novel strategy for esophageal carcinoma.
出处
《郧阳医学院学报》
2004年第5期257-261,269,F002,共7页
Journal of Yunyang Medical College
