| Objective:Research of AML patients with Leukemia stem cells expression, and the effect of arsenic trioxide on the stem cells in the HL 60 cell lines, investigate the speciific surface markers of leukemia stem cells to assess condition, prognosis of value, looking for intervention, new methods for the treatment of Leukemia stem cells.Methods:We use Magnetic-Activated cell sorting method to separate CD34 and CD34/CD 123 positive cells from AML patients bone marrow mononuclear cells. Flow cytometry instrument detection the expression rate of positive cells before and after sorting, analyzed combined with clinical data. Magnetic-Activated cell sorting method to separate CD34 and CD34/CD123 positive cells from the HL-60 cell lines, set different concentration of arsenic trioxide on the group of CD34 cells after 24h, 48h and 72h, use the MTS method to detect the drug to the cell proliferation inhibition.The cells apoptosis were detected with Annexin V-FITC/PI double staining and cell cycle was analyzed with PI single stained in sorted positive cells by flow cytometry.MS-PCR method to detect CD34 positive cells ID4, CDH13, SFRP2 gene methylation, observation of arsenic trioxide to methylation.Results:1.The 20 AML patients Common express with CD34 and CD34/CD123 positive cells, before sorting the expression rate of CD34 positive cells was 32.92±30.16%, the expression rate of CD34/CD123 positive cells was 10.70±12.13%, after using magnetic-activated cell sorting method,the expression rate of CD34 positive cells was 74.87±10.49%, the expression rate of CD34/CD123 positive cells was 68.29± 12.99%; After the separation rate of the expression of CD34 positive cells and CD34/CD123 positive cells expression rate are higher than before (P< 0.05), magnetic-activated cell sorting separation method can effectively improve the purity of positive cells. The ratio obtained of CD34 positive cells was 21.77±8.27%, CD34/CD123 positive cells was 16.35 ± 3.52%.The number of cells was less, causing difficulties for future research.2. The antigen expression rate of CD34 positive cells,initial treatment group was 28.63±25.44%, refractory and relapsed cases was 67.72±22.04%, complete remission group was 4.74±7.64%.The antigen of CD34/CD123 positive co-expression rate of initial treatment group was 10.84±9.51%, refractory and relapsed cases was 23.41±10.01%, complete remission group was 2.91±6.25%. After statistics analysis, the expression of CD34 positive antigen rate of recurrence and relapsed group is higher than initial treatment and complete remission groups, initial treatment group is higher than complete remission group, and lower than the refractory and relapsed cases, two comparative difference was statistically significant (P< 0.05). For the expression of CD34/CD123 positive cells, refractory and relapse cases higher than complete remission group, the difference was statistically significant (P< 0.05), initial treatment group compared with complete remission group, refractory and relapse cases,there was no statistically significant difference (P> 0.05).3. In HL-60 cell lines the expression of CD34 positive populations was 4.06±2.11%, the expression of CD34/CD123 positive populations wasl.65±0.79%; after magnetic activated cell sorted the expression rate of CD34 positive populations was72.86±12.58%, the expression of CD34/CD123 positive populations was 12.67±0.071%.After cultured for a week the expression of CD34 positive populations was 80.71±5.43%, the expression of CD34/CD123 positive populations 11.04±1.25%. The expression of CD34 in positive subset after magnetic activated cell sorted and cultured for a week was higher than HL-60 cell lines, the difference was statistically significant (P< 0.05).The expression of CD34/CD123 in positive subset before and after sorted and cultured for a week was low, there was no statistically significant difference (P> 0.05).4. With different concentrations of As2O3 intervention HL-60 cells and CD34 positive subset after magnetic activated cell sorted,cultured 24h,48h,72h, the proliferation of two groups were obviously inhibition by As2O3 and the highest inhibition rate treated with was 12 umol/1 As2O3 for 72h. The effect showed a dose-and time-dependent manner.5. Most of CD34 positive populations group were in GO/G1 phase (87.7%).Low concentration of As2O3 can induce CD34 positive populations group into the cell cycle phase(S mainly), the number of cells in S and G2/M phase most (51.4±2.80%).When treated with 2 umol/1 As2O3 for 72h, with the increase of the concentration, GO/G1 phase gradually increased, S phase cells increased first,then decreases, G2/M phase without obvious regularity. When treated with 12 umol/1 As2O3 for 72h,mainly block cells in GO/G1 phase (84.21±3.42%)6. AS2O3 could induce the apoptosis and necrosis in the CD34 positive populations group.the effect was significant caused by 12 umol/1 AS2O3 for 72h. The effect showed a dose-and time-dependent manner.As2O3 effect on the CD34 positive populations after magnetic activated cell sorted,regardless of the length of time and drug concentration,cells are given priority to with apoptosis.7. Using methylation specific polymerase chain reaction for to detect the HL-60 cell lines with ID4, CDH13, SFRP2 gene.ID4 gene can be amplified methylated and non methylated bands, decide for methylation positive;CDH13 gene can amplify methylated bands, decide for methylation positive; SFRP2 gene can be amplified methylated and non methylated bands, decision for methylation positive. Different concentrations of AS2O3 effect on the CD34 positive populations, ID4, CDH13 gene methylation still exists.Conclusion:l.The 20 AML patients commonly express with CD34 and CD34/CD123 positive populations, expression rate range from low to high. CD34/CD 123 positive populations expression rate in refractory and relapsed cases were higher than those in complete remission group, in a certain extent can reflect the condition of progress. High expression indicates a poor prognosis, and can thus be used to assess the number and proliferation of the LSC.After magnetic activated cell sorted, the expression of CD34 and CD34/CD 123 positive populations were increased significantly. Immune magnetic activated cell sorting method takes a short, simple operation, of cells with little damage and is effective method to obtain rare leukemia stem cells in the bone marrow of patients with leukemia.2. There were CD34+,CD34/CD 123 positive populations in HL-60 cell lines, accounts for the proportion to be lower, here were CD34,CD34/CD 123 positive populations in HL-60 cell lines, accounts for the proportion to be lower; using magnetic activated cell sorted,the CD34 positive populations expression rate was significantly increased,the CD34/CD 123 positive populations expression rate is still low. After cultured for a week, the CD34 positive populations expression rate was no significant difference,Shows the characteristics of this group is relatively stable. AS2O3 could inhibited proliferation induce the apoptosis and necrosis in the CD34 positive populations group,the effect showed a dose-and time-dependent manner.Most of CD34 positive populations group were in GO/G1 phase.Low concentration of As2O3 can induce CD34 positive populations group into the cell cycle phase(S mainly),high concentration of As2O3 mainly block cells in GO/1l phase.3. The CD34 positive populations of HL-60 cell lines exist ID4, CDH13 and SFRP2 gene methylation positive. As2O3 effect on the CD34 positive populations demethylation effect is not obvious. |
PDF Full Text Request