Quantitative Analysis of Gene Expression for Vascular Endothelial Growth Factor and Its Application.

Vascular endothelial growth factor (VEGF) is a crucial mediator of angiogenesis, and plays an important role in pathogenesis of leukemia. However, the importance of VEGF in differentiation or apoptosis of leukemic cells remains to be evaluated. A competitor DNA fragment template of VEGF gene mimic was constructed with the method of gene recombinant technologies, and a competitive quantitative reverse transcriptase-polymerase chain reaction (cQRT-PCR) for analyzing VEGF gene expression was performed to assess the regulation of VEGF gene expression in the process of all-trans retinoic acid (ATRA)-induced differentiation of an acute promyelocytic leukemia cell line NB4. In construction of a standard curve from which the amount of target cDNA was derived, serial dilutions of the target were co-amplified with a constant amount of mimic, and the intensities of bands corresponding to the target and the mimic were measured. CD11b antigen and nitroblue tetrazolium (NBT) reduction rate of NB4 cells were also assayed at different time points. cQRT-PCR was a sensitive and reliable tool for analysis of VEGF gene expression, with a detectable range from 1 to 2 times 10 the fifth power molecules. The number of VEGF gene transcripts detected by means of cQRT-PCR assay was 42.3, 12.6, 3.6, and less than 1.0 times 10 the fifth powder per microgram of NB4 total RNA at 0, 12, 24 and 48 hours after ATRA treatment, respectively. The rapid down-regulation of VEGF gene expression during ATRA-induced NB4 cell differentiation was accompanied by an upregulation of CD11b expression and an increased NBT reduction rate. In conclusion, cQRT-PCR could be used as an efficient method of qualitative analysis of VEGF gene expression. ATRA significantly depresses VEGF expression and its antileukemic effect can be brought through the two ways of differentiation induction and angiogenesis inhibition.