[The effects of culture supernatant of human GRC-1 cells transfected with PF4 cDNA on the VEGF expression and the growth of ECV304 cells].
OBJECTIVE
To construct the eukaryotic expression vector pcDNA3-PF4-SS, and to detect the effects of the culture supernatant of transfected GRC-1 cells on the VEGF expression in transfected GRC-1 cells and the growth of ECV304 cells.


METHODS
The eukaryotic expression vector pcDNA3-PF4-SS was constructed and identified with Bgl II/BamH I digestion. The pcDNA3-PF4-SS was transfected stably into GRC-1 cells with lipofectamine mediation. The VEGF expression in transfected GRC-1 cells was detected by immunohistochemical staining, and the effect of the culture supernatant of transfected GRC-1 cells on ECV304 cells was detected by MTT colorimetry.


RESULTS
Restrictive enzyme (Bgl II/BamH I)digestion analysis showed that the recombinant expression vector pcDNA3-PF4-SS had been constructed successfully. RT-PCR detection proved that hPF4 cDNA had been transfected into GRC-1 cells. The result of immunohistochemical staining showed that the VEGF expression could be seen in the cytoplasm and on cytomembrane of GRC-1 cells transfected with pcDNA3-PF4-SS, but the expression obviously weakened as comparison with that before transfection. Cell counting and MTT colorimetry manifested that the culture supernatant of transfected GRC-1 cells could inhibit markedly growth of ECV304 cells.


CONCLUSIONS
The eukaryotic expression vector pcDNA3-PF4-SS has been constructed successfully, and stably transfected into the GRC-1 cells. The culture supernatant of transfected GRC-1 cells has obviously inhibitory effect on the growth of ECV304 cells and the VEGF expression in the GRC-1 cells, which lays some foundation for exploring the mechanism for anti-tumor growth and developing tumor vaccine for kidney neoplasms.
Chang
Zheng
Z
Department of Urology, Tangdu Hospitial, Fourth Military Medical University, Xi'an 710038, China. changzheng_612@sina.com
Liu
Fan
F
Han
Hua
H
Sun
Qiang
Q
D002292:Carcinoma, Renal Cell / Q000378:metabolism / Q000473:pathology; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D042783:Endothelial Cells / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007680:Kidney Neoplasms / Q000378:metabolism* / Q000473:pathology; D010978:Platelet Factor 4 / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D042461:Vascular Endothelial Growth Factor A; D010978:Platelet Factor 4