Study On The Differentiation Of K562 Cells Induced By Antisense Oligonucleotide Targeting Survivin

Objective To study the role and mechanism of survivin in differentiation of leukemia cells by antisense oligonucleotide, which will be an important way for gene therapy of leukemia .Methords 1. The protein expression of survivin in k562 cells. For the two groups: k562 cell group and control group (peripheral blood mononuclear cells from normal human), the protein level of survivin was examined by immunohistochemistry(SABC). 2. The morphologic and functional characteristics of k562 cells which were induced by antisense oligonucleotide targeting survivin, and the mechanism of survivin in differentiation of k562 cells.K562 cells were divided into 4 groups: ASON group, NSON group, lipofectin group and control group. 24h and 48h after transfection, the morphologic and ultrastructural changes were observed by light microscope and transmission electron microscope respectively. The function of k562 cells was detected by benzidine dyeing, POX dyeing and NBT reduction test. The average fluorescence density of CD33 and its cell cycle progression were detected by flow cytometry. The protein level of survivin and c-myc was investigated by immunohistochemistry(SABC).Results The protein level of survivin in k562 cells was high while there was no expression in PBMCs. After ASON targeting survivin was transfected, k562 cells were induced to differentiate into erythron and myelocyte according to their morphologic and ultrastructural changes. 24h and 48h after thansfection in ASON group, the positive rate of benzidine dyeing was 11.90 % and 18.20 %, the positive rate of POX dyeing was 17.40% and 29.40%, the positive rate of NBT reduction test was 7.50 % and 12.10 %,which were significantly higher than those of the NSON group, lipofectin group and control group(P<0.05). Meanwhile, the average fluorescence density of CD33 ASON group were 21.43 and 14.86, which was significantly lower than those of the other groups (P<0.05).In ASON group, the cell ratio in G0/G1 phase increased while those in G2/M and S phases decreased. 24h after transfection, the distribution of cell cycle was 37.28% at G0/G1 phase, 16.25% at G2/M phase and 44.87% at S phase. 48h after transfection, the distribution of cell cycle was 45.42 % at G0/G1 phase, 10.88 % at G2/M phase and 40.36% at S phase. The cell cycle progression was significantly inhibited (P<0.05). 24h after thansfection, the protein level of survivin and c-myc in ASON group decreased(P＞0.05),while that remarkably decreased after thansfection for 48h(P<0.05). There was a positive correlation between the protein level of survivin and c-myc(P<0.01).Conclusions　1. The protein level of survivin in k562 cells is high. Survivin may be a new target of leukemia gene therapy. 2. Survivin is related to the differentiation of k562 cells and ASON targeting survivin can induce k562 to differentiate into erythron and myelocyte. 3. The cell cycle progression of k562 cells can be inhibited by ASON targeting survivin with the decline of the protein level of c-myc, which shows that k562 cells are induced to mature differentiation.