Study Of The Expression Of WWOX In Human Chronic Myelogenous Leukemia By Transplanting K562Cells Into Nude Mice

Objective:To explore the biological characteristics of human chronic myelogenous leukemia model Established by different inoculation methods.To investigate the inhibitory effect of Decitabine (DCA)on the growth of xenograft and the expression of WWOX in human chronic myeloid leukemia model in nude mice through different ways of transplantation. Furthermore, to discuss pathogenesis of WWOX gene in human chronic myeloid leukemia.Methods:1. Cultured cell K562of human chronic myelogenous leukemia, then adjusted the concentration of cell to2x107/ml, BALB/c nude mice were divided into four groups(10mice/each group), control group and rest were respectively injected cell of0.2ml via tail vein (iv), by the means of subcutaneous (sc) and intraperitoneal injection (ip). 2. Observed the activity situation and regularly white blood cell count in nude mice. The xenograft model of human leukemia was verified by morphology (peripheral blood, bone marrow and liver and spleen with histopathology), and observed cell morphology changes in blood and bone marrow of leukemia in nude mice. Ratio of CD45positive cells in peripheral blood was analyzed by FCM. RT-PCR was used to detect bcr-ab1mRNA expression of leukemic cells in the Peripheral blood mononuclear cells (PBMCs) in nude mice.3. The model of human chronic myelogenous leukemia in nude mice via subcutaneous was used for drug intervention experiment. Start medication after subcutaneous transplantation tumor diameter to0.5cm. The experimental group (10mice):leukemia model in nude mice was treated with DC A via intraperitoneal, the dose of drug was lmg/kg, was diluted in0.1ml PBS buffer, mice was treated for3weeks (weekly continuous intervention for3days). The control group (10mice): leukemia model in nude mice was treated with0.1ml of saline via intraperitoneal under the equivalent conditions.4. The xenograft growthand white blood cell count in nude mice was observed after the intervention of DC A.5. The mRNAand protein expression of WWOX was detected by nested PCR in PBMCs and xenografts tumor of leukemia mice after the intervention of DCA. 6. Protein expression of WWOX was detected by Western-Blot in PBMCs and xenografts tumor of leukemia mice after the intervention of DCA.Results:1. several leukemic cells can be detected by a microscope on a peripheral blood smear of the group (iv) mice in two weeks after inoculation, and the survival time of these mice was(30.4±2.17)d. Group (sc) mice were inoculated at the subcutaneous and the several tumor nodules can be observed4days after inoculation. The Group (ip) mice, solid tumor and ascites grew faster in abdominal cavity, survival time was (19.6+1.34) d. Comparing the survival time between the three kinds of leukemia model of nude mic, the group (sc) was longer than that of the control A and the groupC, between groups difference was statistically significant (F=198.492, p<0.01). The group (sc) can be observed the subcutaneous transplantation tumor of the earliest, survival time was the longest.2. The peripheral white blood cells increased gradually and emerged a large number of leukemia cells with time of inoculation in the three kinds of established human chronic myelogenous leukemia mice models. A large number of leukemia cells can be detected in bone marrow. Dying nude mice peripheral blood white blood cell count were respectively (29.31±2.58)×109/L,(33.18±2.93)×109/L,(25.48±2.74)×109/L in the three groups, between groups difference was statistically significant (F=14.694, p<0.05). In the three groups of leukemia mice, the CD45positive rates in the peripheral blood were (45.05+3.25)%,(48.55+3.33)%,(35.72+2.54)%, between groups difference was statistically significant (F=35.398, p<0.01). In the three groups of leukemia mice, bcr-ablmRNA expression respectively were0.403±0.165,0.884±0.189,0.924±0.103, between groups difference was statistically significant (F=10.225, p<0.05).3. In Leukemia mice established via subcutaneous, the relative tumor volume (RTV) was(470.42±30.49)mm3in experimental group, while in control group was (580.74±40.63) mm3, RTV was significantly smaller in experimental group treated with DCA than in control group (t=42.137,P<0.05). Observed xenografts tumor cell with hyperchromatic, the tumor cell volume was decreased, and the tumor cell nuclei were hyperchromatic and shrinkage in experimental group. By contrast, the subcutaneous tumor cells of the control group of were arranged in rows and grew actively. White blood cell count treated by DCA increased slowly, and was (15.37±2.56)×109/L. While white blood cell count in the control group increased quickly, and was (22.63±3.41)×109/L. The white blood cell count in the control group was more than thao treated by DCA, and the difference was statistically significant (t=30.21, P<0.01). 4. In Leukemia mice established via subcutaneous, WWOXmRNA expression in PBMCs and xenografts tumor were0.458±0.104、0.618±0.124in the control group, the WWOXmRNA expression treated by DCA for3weeks in PBMCs and xenografts tumor were0.865±1.354、1.265±0.187. The WWOXmRNA expression treated by DC A was higher than that of the control group, and the difference were statistically significant (t=-22.314,P<0.01;t=-28.125,P<0.01).5. In the way of establishing subcutaneous transplantation in leukemia mice, WWOX protein expression in PBMCs and xenografts tumor were relatively low0.354±0.011、0.674±0.010. The WWOX protein expression increased in the experimental group in PBMCs and xenografts tumor, were0.684±0.009、0.903±0.009with the comparison of control group, and the WWOX protein expression level of the experimental group was higher than that in control group, and the difference were statistically significant (t=-451.871, P<0.01; t=-627.142, P<0.01).Conclusion:1. It was a viable way that establishing chronic myelogenous leukemia by transplanting K562cells into nude mice, in Intravenous subcutaneous or intraperitoneal injection. Leukemic manifestation (the subcutaneous tumor) was the earliest, and their surviving time was the longest, and also the number of white blood cell was the most when dying.2. Demethylation drug DC A could upregulate the WWOXmRNA and WWOX protein expression in transplanted tumor tissue of chronic myelogenous leukemia mice.3. DCA could inhibit the growth of K562cells xenografts of chronic myelogenous leukemia in nude mice.