质粒载体介导CD基因对喉癌细胞的杀伤作用研究

目的构建含酵母菌自杀基因CD的质粒表达载体pcDNA3.1(-)CMV.CD,并利用该载体进行喉癌Hep-2细胞株转染研究,体外实验观察前体药物5-FC对稳定表达CD基因的喉癌Hep-2细胞株的杀伤作用.方法通过RT-PCR从酵母菌RNA内扩增出CD基因全长CDS序列,将其定向克隆到质粒表达载体pcDNA3.1(-)CMV中,重组体质粒经XhoI/HindⅢ双酶切鉴定,并对重组体中的CD基因片段进行序列分析,将鉴定好的阳性重组质粒pcDNA3.1(-)CMV.CD运用电穿孔法转入喉癌Hep-2细胞中.经300～600μg/mlG418正筛选14 d和10 μg/ml前体药物5-FC负筛选,获得稳定表达CD基因的喉癌Hep-2细胞株,提取该细胞株细胞的总RNA,经RT-PCR鉴定CD基因的表达.MTT法观察不同浓度5-FC对稳定表达CD基因的喉癌Hep-2细胞及转染空白载体pcDNA3.1(-)CMV的对照组喉癌Hep-2细胞的杀伤作用.结果阳性重组质粒pcDNA3.1(-)CMV.CD经XhoI/HindⅢ双酶切后获得5353bp的片段及496 bp的插入片段,DNA自动序列分析证明重组体质粒含完整的477bp长的CD基因CDS序列.RT-PCR从转染细胞总RNA中扩出453 bp的预期片段.当添加不同浓度的5-FC时,表达CD基因的Hep-2细胞不同程度地被杀死,而对照组的细胞几乎未受到影响,两组细胞的相对生存率具有显著差异性(P<0.05).结论成功构建了质粒表达载体pcDNA3.1(-)CMV.CD,建立了稳定表达酵母菌CD基因的喉癌表达CD基因的Hep-2细胞株,表达CD基因的Hep-2细胞可以被5-FC杀死.

Killing effect of CD gene mediated by plasmid on human laryngeal carcinoma cells

Objective To construct a recombinant plasmid containing the yeast germ suicide gene CD and explore the killing effect of CD gene on Hep-2 cells in combination with 5-FC in vitro. Methods A fragment containing full-lengh coding region of CD was subcloned into expression plasmid pcDNA3.1(-)CMV to construct recombinant plasmid pcDNA3.1(-)CMV. CD identified by enzyme digestion of XhoI/HindⅢ, then the recombinant plasmid was conducted into the Hep-2 cell line by electroporation. Hep-2 cells stably expressing CD was obtained by 14-day positive selection with 300-600 μg/ml G418 and negative selection with 10 μg/ml 5-FC. Total RNA was extracted and the expression of the CD gene in transfected Hep-2 cells was identified by RT-PCR. MTT assay was used to observe the killing effect of 5-FC of different concentration on Hep-2 cells stably expressing CD and the controlled group was Hep-2 cells transfected by pcDNA3.1(-)CMV. Results A fragment of 5353 bp and inserted fragment of 496 bp were got by cutting positive recombinant plasmid of pcDNA3.1(-)CMV.CD with XhoI/Hind Ⅲ. Automatic DNA sequence analysis demonstrated that the recombinant plasmid pcDNA3.1(-)CMV.CD contained integral coding region of CD of 477 bp totally the same as published in GenBank. The expression of CD gene was detected by RT-PCR. The relative survival rate of Hep-2 cells stably expressing CD was significantly lower than those in the control group (P<0.05). Conclusion pcDNA3.1(-)CMV.CD is successfully constructed and CD-expressing Hep-2 cells can be killed by 5-FC, so the recombinant plasmid may be a candidate vector for laryngeal cancer therapy.