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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载体pcDNA3.1(-)CMV.CDglyTK,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-ern-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-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。
Objective To study the treatment of laryngeal cancer by suicide gene CDglyTK. Methods Plasmid expression vector pcDNA3.1 (-) CMV.CDglyTK was constructed by PCR amplification, restriction enzyme digestion, ligation and transformation. The sequence of CDglyTK was analyzed by XhoⅠ / Hind Ⅲ restriction analysis. The Hep-2 cell line stably expressing CDglyTK gene was established, and the expression of CDglyTK gene was identified by RT-PCR and Western-blotting. The inhibitory effect of 5-FC, GCV, 5-FC + GCV on the growth of Hep-2 cells expressing CDglyTK gene was observed by MTT assay. Results Enzyme digestion and sequencing analysis showed that the recombinant plasmid contained complete CD and TK genes. The predicted fragment of 707 bp was amplified from the total RNA of transfected cells by RT-PCR and the 59 kDa protein was detected by West-ern-blotting. The growth of Hep-2 cells expressing CDglyTK gene was inhibited by 5-FC, GCV and 5-FC + GCV. 5-FC combined with GCV had a stronger killing effect. Conclusion CDglyTK fusion suicide gene can be an effective method for gene therapy of laryngeal cancer.