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The purpose of this study was to establish the phospho-specific flow cytometry (phospho-flow) to detect the phosphorylated signaling proteins of leukemia cells and to evaluate its useful value in leukemia study. The bone marrow of leukemia children was collected, and treated by phospho-flow of extracted mononuclear cells (MNC) and phospho-flow of directly fixed bone marrow (BM) respectively. In phospho-flow of extracted MNC, the MNC extracted from BM were fixed and permeabilized, then were cultured with P-AKT and P-ERK1/2, finally were analyzed by flow cytometry. In phospho-flow of directly fixed BM, the BM was treated with fixation/lysis buffer and 90% methanol, then were incubated with the surface CD antibody, P-AKT and P-ERK1/2, finally the treated BM cells were analyzed by flow cytometry. The results showed that the positive rates of P-AKT and P-ERK1/2 in MNC treated by phospho-flow of extracted MNC of 26 leukemia children were 46.2% and 30.8% respectively, while the positive rates of P-AKT and P-ERK1/2 in BM treated by phospho-flow of directly fixed BM were 50.0% and 38.5% respectively. The comparison of positive rates of P-AKT and P-ERK1/2 between the 2 treatment protocol showed no difference (p > 0.05). It is concluded that the phospho-flow of directly fixed BM established by our laboratory can be used to analyze the signaling proteins of leukemia cells.

The aim of study was to investigate the effect of acute lymphoblastic leukemia (ALL) children bone marrow mesenchymal stem cells (MSC) on resistance of K562/A02 cells and its mechanism. MSC obtained from bone marrow of AL children were cultured and identified. The co-culture of MSC and K562/A02 and the culture of K562/A02 cell suspension alone was performed, of which 2 kinds of cells were treated with same concentration of adriamycin (ADM), and the rate of apoptosis was detected by flow cytometry, bcl-2 and bax of K562/A02 were detected by RT-PCR, while mdr1 gene level was detected by FQ-PCR. The results indicated that the MSC separation and proliferation were viable and steady. The apoptosis rate of the K562/A02 cells co-cultured with MSC was 1.97 ± 0.11%, while apoptosis rate of the K562/A02 cells cultured alone was 8.38 ± 0.29%, there was significant difference (p < 0.05). As compared with the K562/A02 cells cultured alone, the bcl-2 gene expression in K562/A02 cells co-cultured with MSC obviously increased; ratio of bcl-2/bax was obviously enhanced. The mdr1 gene level in K562/A02 co-cultured with MSC was no statistical different from K562/A02 cultured alone (p > 0.05), which suggested that adhesion co-cultured with MSC did not induce mdr1 expression higher than the culture of suspension. It is concluded that the MSC of ALL children can escape the leukemia cells from proapoptotic effect of drugs, the resistance of K562/A02 to ADM may be involved in enhancement of bcl-2 gene expression of K562/A02 cells co-cultured with MSC, but not in relation to mdr1 gene in K562/A02 cells themselves.

This study was purposed to investigate the changes of CD4(+) CD25(+) regulatory T cells and NK cells in peripheral blood of acute leukemia children at different stages, the function of immune system and the possible roles of the CD4(+) CD25(+) regulatory T cells as well as NK cells in leukemia immunity. The number and proportion of CD4(+) CD25(+) regulatory T cells and NK cells were detected by flow cytometry in the peripheral blood of 53 acute leukemia children, including 25 patients in new diagnosis and 28 patients in continuous complete remission (CCR), and were compared with that of 20 normal children. The results indicated that the mean proportion of CD4(+) CD25(+) CD127(+) in CD4(+) T cells of peripheral blood in newly diagnosed patients, patients with CCR and normal children were (9.55 +/- 2.41)%, (8.54 +/- 2.51)% and (6.25 +/- 0.85)% respectively, the mean proportions of CD4(+)CD25(+)CD127(+) in newly diagnosed patients and patients with CCR were higher than that in normal children, the mean proportion of CD4(+)CD25(+)CD127(+) in newly diagnosed patients were higher than that in patients with CCR (p < 0.05). At the same time, the NK cell count in patients with acute leukaemia decreased as compared with normal control, while after achieving CCR, the NK cell count in patients were also less than that in normal control (4.11 +/- 3.87% and 10.41 +/- 7.20% vs 14.06 +/- 5.95%, p < 0.05). It is concluded that the application of CD4(+), CD25(+) and CD127(+) to detect regulatory T cells is a simple, reproductive and accurate method, and the CD4(+) CD25(+) CD127(+) T cells can better reflect the proportion of CD4(+)CD25(+) regulatory T cells. The increase of regulatory T cells and decrease of NK cells in pediatric patients with acute leukemia indicate that the function of NK cells may be depressed. Treg T cells play a role in occurrence and development of leukemia, and are involved in down-regulating NK cell function.

This study was aimed to investigate the conditions of culturing in vitro mesenchymal stem cells (MSCs) derived from bone marrow of children with acute leukemia and the biological characteristics of MSCs from leukemia children. The bone marrow MSCs of acute leukemia children were isolated by density gradient centrifugation combined with adherent segregating method and cultured in DMEM/F12. The morphology of Wright stained MSCs was observed under inverted microscope. Cell surface markers were analyzed with flow cytometry. The growth characteristic features of cultured MSCs was measured with MTT method. Induced adipogenic and osteogenic differentiation of MSCs in appropriate induction media was observed. The results indicated that BM-MSCs of acute leukemia children could be successfully cultured in vitro in appropriate conditions. At 24 hours of culture the MSCs began to adhere to wall, grew in colony and appeared in different shapes. As the culture lasted, the MSCs proliferated continuously and shaped in fusiform. After 2 - 3 weeks of culture, MSCs covered the bottom of culture flask. The analysis of growth feature showed that MSCs were in latency for 3 days, and then entered into growth period. After 8 days of culture the growth of MSCs showed to be in plateau stage. The shape of MSCs in 1st and 2nd generation showed to be heterogeneous but the 3rd generation to be homogeneous with long-fusiform. Cells were arranged in shape of whirlpool or radiation. The surface marker analysis showed that the MSCs were positive for CD105, CD29, CD13, but negative for CD34, CD45, CD14 and HLA-DR. The MSCs from leukemia children could be induced into adipocytes and osteocytes in appropriate conditions. It is concluded that (1) MSCs derived from children with acute leukemia can be successfully cultured and passaged in vitro; (2) MSCs from leukemia children not received chemotherapy are more successfully cultured in vitro than those received chemotherapy; (3) the common biological characteristics of MSCs from children with acute leukemia are same as the MSCs from healthy person.

Objective: Primer Express 2.0 software was used to design the primers and the MGB probe. With the plasmid pHaMDR1/A containing mdr1 cDNA as the template, we established a real-time fluorescent quantitative polymerase chain reaction system, which, at the template concentration of 3.061 x 10(3) to 3.061 x 10(9) cps/ml, had a correlation coefficient of 0.988243 between template concentration and threshold cycle value. This PCR method allows sensitive, specific and quantitative detection of human mdr1 gene.

Objective: To investigate the safety and therapeutic effect of low dose (1000 U/m(2)) L-asparaginase (L-Asp) in the treatment of children with acute lymphoblastic leukemia (ALL)., Methods: Six patients were treated with low dose L-Asp after previously suffered severe side effects from standard dose L-Asp (5000 - 10,000 U/m(2)). Twenty-eight blood samples were obtained randomly from 5 of them. Plasma asparagine concentration was detected by reverse phase-high performance liquid chromatography (RP-HPLC)., Results: All the patients treated with low dose L-Asp showed no any toxic symptoms. The plasma asparagine levels in the patients were all above 5 micromol/L except case 4 (4.91 micromol/L) before receiving L-Asp, and were all decreased below 0.5 micromol/L five days after receiving low dose L-Asp, except case 3 (3.70 micromol/L), the results being like that of receiving standard dose L-Asp., Conclusion: Low dose L-Asp has definite efficacy for childhood ALL, while avoids serious side effects from standard dose L-Asp.

To investigate the pattern of human ERMAP gene expression in different cell lines, 15 cell lines derived from hematopoietic tumor, somatic tumor and normal tissue were chosen and were cultured; cells were harvested after culture for 12, 24, 36, 48, 60 and 72 hours; the expression of the human ERMAP was detected by using fluorescent quantitative PCR. The results showed that human ERMAP gene expression was positive in K562 cell line at interval of 12 and 24 hours, and the expression levels were (5.092 +/- 2.331) x 10(6) cps/microl RNA, (5.328 +/- 3.916) x 10(6) cps/microl RNA, respectively; ERMAP gene expression was also positive in ECV304 cell line at interval of 24 hours, and the expression level was (0.84 +/- 0.12) x 10(6) cps/microl RNA; and its expression was negative in other 13 cell lines. It is concluded that human ERMAP gene expression in ECV304 cell line was found first, and its expression in K562 cell line was further confirmed.

In order to investigate the potential of human ERMAP gene in erythroid cell differentiation, K562 cells were induced to erythroid lineage by Ara-C and to macrophage lineage by TPA, human ERMAP mRNA was detected by fluorescent quantitative PCR. The results showed that human ERMAP mRNA increased while K562 cells were induced to erythroid lineage after treatment with Ara-C at 2.5 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. Human ERMAP mRNA not changed while K562 cells were induced to macrophage lineage after treatment with TPA at 2.0 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. It is concluded that human ERMAP gene plays an important role in differentiation and proliferation of erythroid cells.

Objective: To investigate the changes in the activity of Escherichia coli asparaginase (L-asp) and the concentration of asparagines (ASN) in the plasma of the acute lymphoblastic leukemia (ALL) children receiving L-asp containing chemotherapeutic protocol to explore more reasonable usage of L-asp in the treatment of childhood ALL., Methods: L-asp containing hemotherapy regimen of VDLP was used, in which L-asp (10,000 U/m(2)) was administered intravenously every other day for 10 doses in 15 children with ALL. A total of 340 peripheral blood samples were collected at scheduled time points during the therapy and plasma L-asp activity (by spectrophotometric assay) and asparagines concentration (by RP-HPLC) were measured., Results: During the administration of L-asp, the plasma L-asp activity was increasing gradually peaked after eight doses and then decreased gradually, while the plasma concentration of asparagines maintained in complete or nearly complete depletion status. After the therapy courses finished, a plasma L-asp activity above 100 U/L with asparagines almost complete depletion status was lasting for about seven days., Conclusion: The current L-asp containing chemotherapeutic protocols in which L-asp was administered in a dose of 10 000/m(2) intravenously every other day, are efficient enough for the depletion of plasma ASN.