[Human antisense-vascular endothelial growth factor gene therapy for laryngeal tumor by cationic liposome-mediated transfection].
OBJECTIVE
To observe the effect of cationic liposome mediated antisense-vascular endothelial growth factor (VEGF) gene transfection on the growth of laryngeal cancer Hep-2 cells in the nude mice.


METHODS
The VEGF-cDNA gene was cloned by reverse transcriptase polymerase chain reaction (RT-PCR) from human laryngeal cancer, and its eukaryotic expression vector pcDNA3-VEGF (-) with antisense-VEGF gene was constructed and identified by PCR and double-enzyme digestion. The pcDNA3-VEGF (-) was transfected into laryngeal cancer Hep-2 cell line by using cationic liposome (LP 2000). Then, the transfected Hep-2 cells were injected into nude mice and the size of tumor from different groups was observed while establishing laryngeal cancer xenografts in nude mice, and then treating the tumor-bearing mice with liposome-plasmid complex, observing the size of tumor from different groups. The expression of VEGF mRNA in different groups was observed by RT-PCR. The transfected cell ultranstructure was observed by transmission electron microscopy.


RESULTS
The human VEGF-cDNA was successfully cloned and its eukaryotic expression vector with antisense-VEGF pcDNA3-VEGF (-) was constructed. The antisense-VEGF gene was transfected into Hep-2 cell line by using cationic liposome (LP2000). The size of tumor transfected with pcDNA3-VEGF (-) was significantly smaller than that of control groups. While the size of tumor treated with liposome-pcDNA3-VEGF (-) complex was significantly smaller than that of control groups. Many apoptic tumor cells were observed by transmission electron microscopy and the structure of microvessel was also changed. The expression of VEGF mRNA was evidently weaker than that of the control groups.


CONCLUSIONS
The growth of Hep-2 cells could be inhibited significantly by antisense-VEGF gene transfection.
Deng
Zhi-hong
ZH
Department of Otorhinolaryngology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. dengzh@fmmu.edu.cn
Jin
Min
M
Huang
Wei-guo
WG
Qiu
Jian-hua
JH
Liu
Shun-li
SL
Wang
Jin-ling
JL
D000818:Animals; D003001:Cloning, Molecular; D016373:DNA, Antisense; D005260:Female; D015316:Genetic Therapy; D006801:Humans; D007822:Laryngeal Neoplasms / Q000628:therapy*; D008081:Liposomes; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics*
D016373:DNA, Antisense; D008081:Liposomes; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A