ИСТИНА

Cell heterogeneity of tissue specimens accounts for variability in gene expression studies. Thus gene expression data in B-cell lymphomas can be biased by reactive tissue admixture. Hence FACS has a great potential for facilitating molecular techniques such as qPCR. In this work CLL and normal lymphocyte subpopulations were sorted for real-time qPCR to determine the expression levels of BCR pathway molecules CD79a, Lyn, Syk and Zap-70. Sorting was performed on FACSAria II. CLL cells were purified from blood samples (n=20) based on CD5+/CD19+/CD23+/CD38+- immunophenotype. Normal CD5+B-cells with CD5+/CD19+/CD23-/CD38+ phenotype were purified from human tonsils (n=10) where they comprised ~10% lymphocytes. Peripheral blood B- (n=10) and T-cell (n=10) samples were sorted based on CD19+/CD45+ and CD3+/CD45+ phenotypes respectively. Over 104 cells were sorted per specimen; RNA and cDNA were further obtained. Expression levels of CD79a, Syk, Lyn, Zap-70 were assessed by real-time qPCR and normalized based on 3 reference genes (YWHAZ, UBC, HPRT1). Median gene expression levels were compared for CLL, normal CD5+B-cells, normal CD5-B-cells and normal T-cells. T-cells differed significantly from all groups of B-cells (Mann-Whitney test, p<0,05) and served as negative control for BCR-associated kinases Syk, Lyn, CD79a and positive control for T-cell kinase Zap-70. Significant differences between malignant and normal B-cells were obtained for Syk (CLL vs. CD5- B-cells; CLL vs. CD5+B-cells), Lyn (CLL vs. CD5+B-cells) and Zap-70 (CLL vs. CD5- B-cells): Syk and Lyn were downregulated in malignant cells, Zap-70 was upregulated. No significant difference was obtained for CD5- vs. CD5+ normal B-cells. CD5+ and CD5-subpopulations of normal B-cells don’t exhibit any difference in BCR pathway gene expression. CLL cells have decreased levels of BCR-associated molecules Syk and Lyn and increased Zap-70 compared to normal B-cells, which may contribute to signaling impairment in CLL.