Clinical And Experimental Study On The Treatment Of Myeloid Malignancies By Dacitabine

Objective: Clinical research by different doses of decitabine application evaluation of curative effect of hematologic malignancies(AML/MDS) in patients with, adverse reactions, peripheral blood recovery time, survival time and other indicators for choosing more appropriate dosage regimen; Experimental study on decitabine on leukemia K562 cell line and normal mononuclear cells, to investigate the mechanism of Decitabine on leukemia cells and provide a theoretical basis for further application in leukemia.1. Study subjects and methods:1.1 Patients with acute myeloid leukemia(AML) or bone marrow hyperplasia syndrome(MDS) from May 2010 to December 2015 who used Decitabine treatment were collected.According to different dosage groups respectively for two groups of patients, age, gender,risk classification, the response rate after 1-2 courses of decitabine, to the drug adverse reactions(bone marrow suppression, infection, bleeding, liver or kidney damage),peripheral blood recovery time, survival time were statistically analyzed.1.2 Decitabine was donated to Xinshidai Pharmaceutical Companies in Shandong,leukemia K562 cells subcultured by the Blood Medicine Laboratory of Shaanxi Provincial People’s Hospital. Using an inverted microscope observate K562 cells’ morphology in different time(24h, 48 h, 72h) when the same concentration of Decitabine(100 μmol/L) act on; Trypan blue staining method tests the K562 cells’ viability of different concentration(0 μ mol / L, 50 μ mol / L, 100 μ mol / L) for different time(24h, 48 h, 72h). Flow cytometry tests the K562 cells of early and late apoptotic rates in different drug concentration at different time(24h, 48h). Testing the effects of different concentrations of Decitabine on K562 cells on the expression of BCR/ABL fusion gene P210 protein by real-time fluorescence quantitative PCR method in a certain time range. Collect three copies of normal human peripheral blood, separated by Ficolldensity gradient centrifugation to obtain mononuclear cells, select 100 μmol/L,decitabine’s working congcentration, to worked on normal mononuclear cells for 72 h,Trypan blue exclusion staining was used to detect the mononuclear cells’ proliferation of drug group and control group. The effect of single drug and combination of Ara-C on the activity of Caspase-3 was detected by Western blotting.2. Experimental results:2.1 The constituent ratioes of two groups of patients with the different dose regimens(age,gender, risk classification) were analyzed, and the data were comparable.(1) The group of 20 mg/㎡/d1-d5 is more effective than the group of 15 mg/㎡/ d1-d5(P< 0.05).(2) The rate of 20 mg/㎡/ d1-d5 group’s adverse reactions are higher than 15 mg/㎡/d1-d5 group’s in bone marrow suppression and infection(P<0.05).(3) 15 mg/㎡/ d1-d5 group: the duration WBC<4.0×109 / L were 10 ~ 33 days, with an average 18.90 days; the duration PLT 50×109/L was 12-35 days, with an average of 22 days.(4) The statistical 20 mg/㎡/d1-d5 group in a survival(11 months) and 15 mg/㎡/d1-d5 group in a survival(15 months) showed no statistically significant difference(P> 0.05).2.2(1) Observating the K562 cells’ morphological changes at different time under inverted microscope: the same concentration of Decitabine(100 μmol/L) in K562 cells after 24 h and the integrity of cell morphology showed no obvious abnormalities.After 48 h the cells swelled, defored and, appaired apoptotic bodies occasionally; After72 h the cells number decreased, the shape become more irregular and more apoptotic bodies and cell debris, some cells appeared bubble deformation.(2) Trypan blue staining tests the effects of decitabine on inhibing K562 cells proliferation: different concentrations of Decitabine(50 μ mol / L, 100 μ mol / L) at 24 h,48h and 72 h were inhibited the proliferation of K562 cells, and the inhibitory action in a time and concentration dependent manner.(3) To test the K562 cells’ apoptosis Decitabine effects on using Annexin V-7AAD double labeling method of FCM: comparing the cells’ apoptotic rates of different concentration of decitabine treatment cells in the same time measurement. Experimental group(50μ mol /L, 100μ mol /L) and the control group apoptosis compared as well as the apoptosis between experimental groups(50 μmol/L, 100 μmol/L) after 48 h were statistical difference(P<0.05), and after 24 h in the experimental group(50 μmol /L, 100μmol /L) between difference was not statistically significant(P>0.05). Compared the same concentration of decitabine treated cells the apoptotic rate of experimental group(50 μmol/L, 100 μmol/L) after different time(24h and 48h), the differences were statistically significant(P < 0.05).(4) Real-time fluorescence quantitative PCR were detected in K562 cells of BCR/ABL fusion gene P210 protein expression: different concentrations(50 μmol/L, 100 μmol/L)of Decitabine role in K562 cells for 72 h and detected by real-time quantitative PCR. The results show that the Decitabine can cut K562 cells’ BCR / ABL fusion gene P210expression(P < 0.05) and the down-regulation of gradually increased with the concentration of Decitabine increases.(5) Trypan blue exclusion tests the effect of Decitabine on normal mononuclear cells:the same concentration of Decitabine(100 μmol/L) effected on normal mononuclear cells at different time(24h, 48 h, 72h) by Taiwan trypan blue staining of living cells count and found that decitabine proliferation of normal mononuclear cells without obvious effect(P > 0.05).(6) Test the effect of decitabine on caspase-3 activity using Western blot: the expression of cleaved caspase-3 was not obvious in control group, while the experimental groups’ expression levels were significantly higher than those in the control group;decitabine(100 μmol/L) group of cleaved caspase-3 expression level was significantly higher than that of decitabine group(50 μmol/L).3. Conclusion:The group of 20 mg/ ㎡ /d1-d5 is more effective than the group of 15 mg/ ㎡ /d1-d5 though the median survival time was no difference, at the same time Decitabine adverse reactions such as bone marrow suppression and infection rate of 20 mg/ ㎡ / d1-d5 scheme were compared to 15 mg/ ㎡ / d1-d5 scheme’s is higher.The growth and proliferation of K562 cells were significantly inhibited by Decitabine, and the inhibition intensity was concentration and time dependent; The expression of BCR/ABL fusion gene P210 expression in K562 cells was significantly down regulated, and the Caspase-3activity was significantly increased.