融合自杀基因CDglyTK治疗喉癌的体外实验研究

目的 研究融合自杀基因CDglyTK治疗喉癌。方法 PCR扩增、酶切、连接、转化等构建质粒表达栽体pcDNA3．1（-）CMVCDglyTK，XhoⅠ／HindⅢ酶切鉴定，测序分析CDglyTK基因序列。建立稳定表达CDglyTK基因的Hep-2细胞株，RT—PcR及Western-blotting鉴定CDglyTK基因的表达。MTT法观察5-FC、GCV、5-FC＋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。West—era-blotting检测到该基因表达的59kDa的蛋白。表达CDglyTK基因的Hep-2细胞在5一FC、GCV、5-FC＋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。

Study of treating laryngeal cancer with suicide fusion gene CDglyTK in vitro

Objective] To study the in vitro treatment of laryngeal cancer using suicide gene CDglyTK. Methods] Plasmid pcDNA3.1(-)CMV.CDglyTK, constructed by PCR, enzyme digestion, ligation and transduction etc was verified by digesting 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 μg/mL) for 14 days. The expression of CDglyTK mRNA in transfected Hep-2 cells was tested by RT-PCR.In vitro chemosensitivity of CDglyTK -expressing Hep-2 cells to 5-FC, GCV or 5-FC +GCV was detected by MTT assay. Results] 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 kDa 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.