[VEGF]hTERT复合调控序列介导HSV-tk自杀基因诱导肿瘤细胞的凋亡

Tumor Cell Apoptosis Induced by HSV-tk Suicide Gene in Mediation of VEGF hTERT Multiple Regulatory Sequence

目的构建人端粒酶催化亚单位(hTERT)核心启动子及血管内皮生长因子(VEGF)增强子复合调控序列的真核表达载体,并检测在复合调控序列调控下HSV-tk自杀基因对肺癌细胞A549和肝癌细胞SMMC-7721凋亡的影响。方法构建复合调控序列[VEGF]hTERT和胸苷激酶(tk)基因的真核表达载体pcDNA3.1(+)-[VEGF]hTERT-tk和PCMV启动子的真核对照表达载体pcDNA3.1(+)-tk,转染肺癌细胞A549和肝癌细胞SMMC-7721,G418筛选抗性克隆,给入前药丙氧鸟苷(GCV),MTT比色法测定细胞生长抑制率,流式细胞仪检测肿瘤细胞凋亡率。结果质粒pcDNA3.1(+)-[VEGF]hTERT-tk和pcDNA3.1(+)-tk经双酶切及PCR鉴定,证明构建正确。转染了带有不同启动子的真核表达载体的肿瘤细胞,在给入前药GCV后均能表现出对细胞生长的抑制作用,GCV对A549细胞的抑制率大于SMMC-7721细胞,并能引起肿瘤细胞凋亡。[VEGF]hTERT在A549细胞中的活性较在SMMC-7721中高。结论HSV-tk自杀基因在肿瘤细胞内瞬时表达即能引起靶细胞凋亡,且复合调控序列在肿瘤细胞中的活性较通用真核细胞转录启动子PCMV高,为深入进行自杀基因治疗的研究提供了一定理论依据。

Objective To construct a eukaryotic expression vector for the multiple regulatory sequence consisting of a promoter of human telomerase reverse transcriptase（hTERT）and an enhancer of vascular endothelial growth factor（VEGF）, and explore the influence of HSV- tk suicide gene on the apoptosis of lung cancer A549 cells and liver cancer SMMC-7721 cells in mediation of the multiple regulatory sequence. Methods Construct eukaryofic expression vectors pcDNA3.1 （ ＋ ）-[VEGF]hTERT-tk and pcDNA3.1 （ ＋ ）-tk, containing multiple regulatory sequence[ VEGF] hTERT and PCMV promoter respectively. Transfect A549 and SMMC-7721 cells with pcDNA3.1 （ ＋ ）-[ VEGF] hTERT-tk, using pcDNA3. 1 （ ＋ ）-tk as control.Screen Neor clones with G418 and treat with gancyclovir（GCV）.Determine the inhibitory rate of cell growth by MTT method and the tumor cell apoptosis rate by flow cytometry. Results Restriction analysis and PCR proved that recombinant plasmids pcDNA3.1（ ＋ ）-[VEGF]hTERT-tk and pcDNA3.1（ ＋ ）-tk were constructed correctly. GCV inhibited the growth and induced the apoptosis of tumors cells transformed with both pcDNA3.1（ ＋ ）-[VEGF]hTERT-tk and pcDNA3.1（ ＋ ）-tk. However,the inhibitory rate of A549 cells was significantly higher than that of SMMC-7721 cells ,and the activity of [VEGF]hTERT in A549 cells was significantly higher than that in SMMC-7721 cells. Conclusion The transient expression of HSV-tk suicide gene in tumor cells induced the apoptosis of target cells, and the activity of multiple regulatory sequence in tumor cells was higher than that of universal eukaryote transcriptional promoter PCMV. It provided a theoretical basis for further study on suicide gene therapy.