Investigation Of Expression Of Vascular Endothelial Growth Factor In Acute Leukemic Cells

IntroductionIn recent investigations, the role of angiogenesis in the growth and metastasis of solid tumors has been clearly established. In recent years increased angiogenesis has been reported in the bone marrow of patients with hematological malignancies, such as acute myeloid leukemia (AML) , acute lymphocytic leukemia (ALL) , multiple myeloma (MM). . . etc.Vascular endothelium growth factor ( VEGF) is currently considered the most relevant and the most endothelium specific of the already known angiogenic growth factors. It can alter micro vascular permeability , and stimulate endothelial cell migration and proliferation. A trial of cell culture in vitro demonstrated that some cell lines of hematological malignancies expressed VEGF and it's receptors. Fiedler et al also found VEGF transcription in a high percentage of patients with AML. To investigate whether expression levels of VEGF in acute leu-kemic cells are increased and whether VEGF play an important role in the leukemogenic process, we detected the expression levels of VEGF in bone marrow mononuclear cells from patients with newly diagnosed AML on protein level using immunocytochemistry and flow cytometry (FCM).Materials and methods1. Clinical cases59 patients with newly diagnosed AML were detected, among them 31 were AML, 28 were ALL; average age was 40.5(15 -75). 14 cases with normal bone marrow were used as normal control.2. Isolation of mononuclear cells3 - 5ml heparinized blood or bone marrow liquor was separated by density gradient centrifugation. These preparations contained at least 90% blast cells.3. Detecting VEGF expression by immunocytochemistryWe examined the VEGF expression status using SAP method. The sample was judged positive when percentage of positive cell > 20% , whose cytoplasm were dyed red. Replacement primary antibody with PBS was used as negative control.4. Detecting VEGF expression by flow cytometry(FCM)The samples of 30 patients with acute leukemia and 9 control cases were examined using FCM. Mononuclear cells were fixed with 70% cold alcohol, and then incubated with anti ?VEGF polyclonal antibody and FITC labeled secondly antibody in turn. Replacement primary antibody with PBS was used as negative control. The expression levels of VEGF were expressed as mean fluorescence intensity (MFI).5. Cell proliferation period analysis by flow cytometry Leukemic cells fixed in 70% alcohol were dyed with PI followingRNAase treatment. Cells ' proliferation periods were analyzed by FCM, and the results were expressed as proliferation index (PI).6. Statistical methods; Statistical analysis were performed usingstatistical SPSS software, x2 test? nonparametic Mann - Wilcoxon test, linear regression were used.Results1. VEGF expression detected by immunocytochemistry.VEGF positive rate was significantly higher in acute leukemia group than that in control group [52. 5% (31/59) vs. 14. 3% (2/ 14) ] , P < 0. 01, and was higher in AML group than that in ALL group [67.7% (21/31) vs. 35.7% (10/28) ], P<0.05.2. VEGF expression detected by Flow cytometryVEGF MFI representing VEGF expression levels were significantly higher in patients with acute leukemia (median; 2.045, P25 - P75: 0.553 -4.965) compared with control patients (median; 0.000, P25 -P75: 0.000-0.865), z=3.113, p<0.01. VEGF MFI were lightly higher in patients with AML (median; 3.230, P25 - P75: 0. 580 -4. 290) compared with patients with ALL (median; 3.026, P25 - P75: 0.000- 7.290).The results obtained by immunocytochemistry and FCM are consistent.VEGF MFI were logarithmically correlated with leukemic cells' PI, PI=23.2642.7831nMFI, r=0.448, P<0.01.DiscussionAngiogenesis is an obligatory event connected with tumor progression in the form of growth, invasion and metastasis. When a solid tumor mass grow to 1 - 2mm3, it will remain stillness or degenerate without new microvessel. On the one hand new microvessel can promote tumor growth via providing oxygen and nutrition for tumor. On...