摘要
目的:研究融合自杀基因CDglyTK联合前体药物对喉癌Hep-2细胞的杀伤效应。方法:构建质粒表达载体pcDNA3.1(-)CMV.CDglyTK,XhoⅠ/HindⅢ酶切鉴定,测序分析CDglyTK基因序列。电穿孔法将重组质粒转染Hep-2细胞,400mg/LG418筛选14d获得稳定表达CDglyTK基因的Hep-2细胞,RT-PCR及Western-blotting鉴定CDglyTK基因的表达。以转染空白载体pcDNA3.1(-)的Hep-2细胞为对照,MTT法观察5-FC、GCV、5-FC加GCV对表达CDglyTK基因的Hep-2细胞生长的抑制作用。结果:酶切和基因测序分析证明重组质粒含完整的CD及TK基因,RT-PCR从转染细胞总RNA中扩出707bp的预期片段,Western-blotting检测到该基因表达的分子量为59000的蛋白。表达CDglyTK基因的Hep-2细胞在5-FC、GCV、5-FC加GCV干预下生长受到抑制,5-FC与GCV联合有更强的杀伤效应。结论:CDglyTK融合自杀基因可以成为基因治疗喉癌的有效方法。
Objective：To study the killing effect of suicide gene CDglyTK combined with GCV or 5-FC on the human laryngeal carcinoma Hep-2 cell line in vitro. Method.-Constructed plasmid pcDNA3.1 （-）CMV. CDglyTK was verified by enzyme digestion of Xho Ⅰ/Hind Ⅲ and automatic sequence analysis, then it was introduced into Hep-2 cells by electroporation to yield cells expressing CDglyTK stably after selecting with G418（400 ng/L） for 14 da. The expression of CDglyTK mRNA in transfected Hep-2 cells was tested by RT-PCR. Compared with Hep- 2 cells transferred with pcDNA3.1 （-）, in vitro chemosensitivity of CDglyTK -expressing Hep-2 cells to 5-FC , GCV or 5-FC ＋GCV was detected by MTT assay. Result：The recombinant plasmid contained full-length coding region sequence of CD and TK gene. A anticipated 707 bp fragment was amplified from total RNA of CDglyTK- expressing Hep-2 cells by RT-PCR and a fusion protein of 59 000 was detected in cell extract from transfected Hep-2 cells. In vitro study growth of CDglyTK-positive Hep-2 cells were inhibited by 5-FC,GCV or 5-FC ＋GCV respectively,and the antitumour effect of 5-FC ＋GCV is superior to 5-FC or GCV. Conclusion：CDglyTK may be a candidate for treating human laryngeal cancer.
出处
《临床耳鼻咽喉头颈外科杂志》
CAS
CSCD
北大核心
2007年第12期555-558,共4页
Journal of Clinical Otorhinolaryngology Head And Neck Surgery
基金
国家自然科学基金资助项目(No:30300414)
