[Construction of c-fms antisense eukaryotic expressing vector bearing AFP enhancer and its clinical significance].
OBJECTIVE
To construct human c-fms antisense eukaryotic expressing vector that would express in human hepatoma cells efficiently and specifically and to observe its affection on biological behavior of hepatoma cells.


METHODS
A c-fms fragment centered by TAC sequence coding for 571(st) tyrosine of CSF-1R was amplified with PCR. The fragment was cloned inversely into pcDNA(3) vector (constructed plasmid was named as 'pAS'). Amplified human AFP enhancer core region fragment was cloned into pAS (constructed plasmid was named as pAEAS). PcDNA(3), pAS and pAEAS were transfected into HepG(2) hepatoma cell line and HeLa cervical carcinoma cell line with calcium phosphate method, respectively. Growth rate and apoptosis of transfected cells were observed.


RESULTS
Detected sequences of human c-fms antisense fragment and AFP enhancer core region fragment were both consistent with the sequences registered in Genbank. The growth rates of HepG(2) cells transfected with antisense gene were slower than control (P<0.05). The inhibitory effect of pAEAS was more remarkable than that of pAS (P<0.05). Apoptoic rates of pcDNA(3), pAS and pAEAS group cells in HepG(2) cells were 5.25%, 14.7% and 31.2%, respectively (P<0.01). Apoptoic DNA ladder was observed in pAEAS group. In HeLa cell groups, growth rates of cells in pAS and pAEAS groups were slower than control, while no difference was observed between them (P>0.05). Apoptoic rates of pcDNA(3), pAS and pAEAS group cells in HeLa cells were 3.99%, 8.27% and 8.66%, respectively (P<0.05). No apoptoic DNA ladder was observed in HeLa cells.


CONCLUSIONS
Human c-fms antisense eukaryotic expressing vector bearing AFP enhancer selectively inhibit the growth of AFP positive hepatoma cells. It enables to induce apoptosis of hepatoma cells and is a new gene therapy method for hepatocellular carcinoma.
Cui
J
J
Department of Gastroenterology, Zhujiang Hospital, First Military Medical University, Guangzhou 510282, China.
Yang
D
D
D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology*; D002455:Cell Division / Q000187:drug effects; D016373:DNA, Antisense / Q000235:genetics / Q000494:pharmacology*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000037:antagonists & inhibitors* / Q000235:genetics; D014162:Transfection; D019076:Transgenes; D014407:Tumor Cells, Cultured; D000509:alpha-Fetoproteins / Q000235:genetics
D016373:DNA, Antisense; D000509:alpha-Fetoproteins; D016186:Receptor, Macrophage Colony-Stimulating Factor