Effect of Notch Signaling Pathway on Differentiation of Mouse Embryonic Stem Cells into Hematopoietic Stem Cells or Hematopoietic Progenitor Cells by VEGF / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of Notch signaling pathways on differentiation of mouse embryonic stem cells(ESC) into haematopoietic stem cells or haematopoietic progenitors cells(HSC/HPC).@*METHODS@#Mouse embryonic stem cells were proliferated in vitro to form embryoid bodies; the differentiation of embryoid bodies should be induced in vitro, the experiments were divided into BE, control, VEGF, DAPT and VEGF-DAPT groups; HSC/HPC ohenotype: CD117D34Sca1 was detected by flow cytometry; the related gene expression was detected by RT-PCR.@*RESULTS@#The number of VEGF-induced HSC/HPC in VEGF group was significantey higher than that in the control and EB group (P<0.05), suggesting that VEGF promotes ESC differentiation to HSC/HPC; the number of DAPT-induced HSC/HPC in DAPT group was significanty higher than that in the Control and EB groups(P<0.05), suggesting that DAPT promotes ESC differentiation to HSC/HPC; the number of VEGF+DAPT-induced HSC/HPC in VEGF-DAPT group was significantly higher than that in VEGF and DAPT groups(P<0.05), suggesting that DAPT and VEGF play a synergistic role to promote differentiation of ESC into HSC/HPC.@*CONCLUSION@#Notch signal pathway inhibits differentiation of ESC into HSC / HPC by VEGF.

Analysis of the key factors in SCID mouse-human leukemia model / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was to explore the key factors in leukemia model through the analysis of mouse with bad life state in the modeling process of leukemia so as to provide the theoretical reference for improving the success rate of modeling.</p><p><b>METHODS</b>At 1 week after inoculation of leukemia cells into SCID mice, the life status and peripheral hemogram of SCID mice were tested, the bone marrow smears, splean biopsy and spleen index of mice were examined after dissecting mormal and agoned/died mice during modoling, and the examined results were compared.</p><p><b>RESULTS</b>As compared with control mice, the life status of experimental mice was poor; the blood smear test showed juvenile cells, slightly more white blood cells with irregular shape and partial rupture, the lymphocytes and band cells obviously increased, the neutrophile granulocytes showed nuclear left shift; the bone marrow smears showed larger cell volume, smaller mulcoplasm, abnormal morphology of cells and cell nuclei and serious cell rapture; the spleen examination showed that the spleen diplayed enlargement and hyperemia to varying degree, the spleen index obviously increase, the spleen interstitial expansion, cell disordered arragement and irregular cell shope were observed, however there was no infiltration of leukemia cells in control and experimental mice.</p><p><b>CONCLUSION</b>The mouse age, pathway of inoculating the leukemia cells, sterile condition in breading and avoiding the rejection and inflammatory response in modeling process are the key foctors influencing the modeling success.</p>

Effect of notch signaling pathway on VEGF promoting rat mesenchymal stem cell proliferation / 中国实验血液学杂志

This study was purposed to investigate the effect of Notch signaling pathway on VEGF promoting the proliferation of rat mesenchymal stem cells (MSC). Rat MSC were cultured in vitro, and the cells in logarithmic growth phase were used for experiments. The inhibitor DAPT was used to block Notch signaling pathway, and the effect of the pathway on VEGF promoting proliferation of MSC was observed. The experiment was divided into 4 groups: control, VEGF, DAPT and VEGF+DAPT. The CCK-8 was used to assay the cells proliferation of each group, while RT-PCR was used to detect the changes of related genes (Notch1, Notch2, Flk-1, Hes-1) at mRNA levels. The results indicated that the cells survival rate MSC in DAPT group and VEGF+DAPT group was low in each time point (24 h, 48 h, 72 h), the cell number decreased, and the cells became rounded. The survival rate of MSC in VEGF group was the highest; the difference of cell survival rate was statistically significant between the groups (P < 0.01); Compared with the control group, the mRNA expression level of Notch1, Notch2 and Flk-1 in VEGF group was raised, while the expression level of Notch1 and Notch2 in DAPT group and VEGF+DAPT group come down, with statistically significant differences (P < 0.05); whereas the mRNA expression level of Hes-1 in VEGF group was down-regulated, but that in DAPT group and VEGF+DAPT group was up-regulated, and the difference was statistically significant (P < 0.05). Flk-1 mRNA level in DAPT group and VEGF+DAPT group was slightly lower, but the difference was not statistically significant (P > 0.05). It is concluded that Notch signaling pathway plays an important role in promoting the proliferation of rat MSC, treated with VEGF, however, the DAPT can weaken this effect.

Level of SDF-1/CXCR4 in children with acute leukemia and its significance / 中国实验血液学杂志

The objective of this study was to detect the level of plasma stromal cell-derived factor-1 (SDF-1) and the expression of CXCR4 (SDF-1 receptor in bone marrow cells) in children with Acute Leukemia (AL) and to investigate the relationship between the expression of CXCR4 and extramedullary infiltration. 48 children with acute leukemia and 20 with non-hematologic malignancies were selected into the AL group and the control group respectively. The peripheral plasma and bone marrow cells were collected. The level of SDF-1 in peripheral plasma was detected by ELISA and the expression of CXCR4 in bone marrow cells was determined by flow cytometry. The results showed that the levels of SDF-1 in peripheral plasma and the expression of CXCR4 in bone marrow cells of AL group was significantly higher than that of control group, among which the level of SDF-1 of the acute lymphoblastic leukemia (ALL) group was also higher than that of the acute myeloid leukemia (AML) group, the expression level of CXCR4 in the bone marrow cells of the extramedullary infiltration (EI) group was higher than that of the non-extramedullary Infiltration (NI) group, and all the differences between the both groups were significant. It is concluded that SDF-1 and CXCR4 express a high level in children with AL, which closely relates with the type of leukemia and the migration and infiltration of leukemia cells in bone marrow.

Effects of PTK787 on cell proliferation and expression of fak mRNA in K562 / 中国实验血液学杂志

The aim of this study was to investigate the effects of tyrosine kinase inhibitor PTK787 on cell proliferation, cell cycle and the expression of fak mRNA of human chronic myeloid leukemia (CML) cell line K562, and to explore the mechanism of PTK787 against acute myeloid leukemia. The MTT method was used to detect the effects of PTK787 in various concentrations and at different time points on proliferation of K562 cells; the flow cytometry was used to determine the effects of PTK787 in different concentrations on cell cycle of K562 cells; the RT-PCR was used to assay the expression of fak mRNA in K562 cells treated with PTK787 for 48 hours. The results showed that along with increasing of the concentration and prolonging of time, the inhibitory rate of PTK787 on K562 proliferation was gradually enhanced. The comparison between various concentration groups at same time or comparison between various time groups in same concentration showed significant differences (p < 0.05), in which the effect of 320 micromol/L PTK787 on cells was strongest, while the continuous increase of PTK787 concentration or prolong of action time did not enhance the inhibitory rate on K562 proliferation. With increasing of drug concentration, the cell proportion in G(1) phase gradually increased, the cell proportion in S phase gradually decreased, the comparison between various groups revealed significant differences (p < 0.05), however the continuous increase of drug concentration from 160 micromol/L did not obviously change the cell proportion in phases of cell cycle. With increasing of drug concentration, the expression of fak mRNA in K562 cells gradually reduced with significant differences between various groups (p < 0.05), but with continuous increase of drug concentration from 160 micromol/L, the effect of PTK787 on the expression of fak mRNA in K562 cells also did not obviously change. It is concluded that the PTK787 shows effect of anti-leukemia cells through inhibiting transformation of the K562 cells from G(1) phase into S phase and decreasing the expression of fak mRNA in cells.

Dynamic analysis of expression of VEGF and its receptor-2 in mouse model with acute myeloid leukemia / 中国实验血液学杂志

The objective of study was to explore the role of vascular endothelial growth factor (VEGF) and its receptor-2 in pathogenesis of acute myeloid leukemia. The acute myeloid leukemia model was established on 20 mice with severe combined immunodeficiency (SCID) transplanted by HL-60 cells. The mice were divided into the normal control and test group randomly. The expression of VEGF was detected by enzyme linked immunosorbent assay (ELISA). The expression of VEGFR-2 mRNA was detected by RT-PCR. The results showed that the establishment of acute myeloid leukemia model was succeeded on all SCID mice by HL-60 cell transplantation. The expressions of VEGF and VEGFR-2 mRNAs could be determined on all mice. As compared with the normal control group, the expressions of VEGF and VEGFR-2 mRNAs in the test group significantly increased, but gradually increased during the course of disease. It is concluded that the abnormal expressions of VEGF and VEGFR-2 exist in mice with acute myeloid leukemia, which may be involved in the pathogenesis of AML.

Effect of fas mRNA expression on cytochrome C-induced apoptosis of HL-60 cells in vitro / 中国实验血液学杂志

The aim of this study was to investigate the effect of cytochrome C on apoptosis of HL-60 cells and to explore the mechanism of HL-60 cell apoptosis induced by cytochrome C. The HL-60 cells were treated with different concentrations of cytochrome C for 24 hours. The concentrations of cytochrome C were as follows: 0 (control group), 9.375 mg/L, 18.75 mg/L, 37.5 mg/L, 75 mg/L and 150 mg/L. The apoptosis was analyzed by flow cytometry (FCM) after HL-60 cells were treated with different concentrations of cytochrome C for 24 hours. The fas mRNA expression changes of relevant apoptotic genes were examined by reverse transcription-polymerase chain reaction (RT-PCR) after HL-60 cells were treated with different concentrations of cytochrome C for 24 hours and were analyzed half-quantitatively. The expressions of fas involved in HL-60 treated with different concentrations of cytochrome C for 24 hours were detected by Western blot. The results indicated that the flow cytometry (FCM) (PI straining) showed obvious hypo-diploid peak before G(1) phase in the treated group, and its apoptotic ratio increased in a dose-dependent manner. The fas mRNA expression levels of the relevant apoptotic genes could be detected by RT-PCR after HL-60 cells treated with different concentrations of cytochrome C for 24 hours, and the expression of fas mRNA in HL-60 cells was increased by cytochrome C in dose-dependent manner within range of 0-37.5 mg/L. It is concluded that the cytochrome C up-regulates expressions of fas mRNA and their protein so as to induce apoptosis of the HL-60 cells.

Influence of cytochrome C on apoptosis induced by daunorubicine in acute myeloid leukemia (AML) cells / 中国实验血液学杂志

The purpose was to study the responses of AML cell treated with cytochrome C and to explore the influence of cytochrome C on apoptosis of AML cell induced by daunorudicine (DNR). The differentiation of AML cell was detected by Wright-Giemsa staining and NBT test, the apoptosis of AML cell was assayed by flow cytometry and fluorescence microscopy. The results showed as follows: (1) different concentrations of cytochrome C could induce different effects on AML cells. Concentration of cytochrome C for differentiation was 10 microl/ml, for apoptosis was 20 microl/ml, and for necrosis was 40 microl/ml. (2) the apoptosis of AML cells decreased with the administration of cytochrome C in 10.0 microg/ml before treating AML cells with DNR (P < 0.01), but no change was shown with the administration of cytochrome C in 20.0 microg/ml (P > 0.05). (3) in reverse sequence, administrating of cytochrome C in 10 microl/ml and 20 microl/ml after treating AML cells with DNR, two different concentrations of cytochrome C could increase the apoptosis of AML cells (P < 0.01). It is suggested that cytochrome C may probably affect the apoptosis of AML cells induced by DNR.

Effect of cytochrome C on HL-60 cell apoptosis and its relationship with the relevant genes bcl-2 and bax / 中国实验血液学杂志

To study the effect of cytochrome C on HL-60 cells in vitro and the mechanism of expression changes of relevant apoptotic genes, the inhibition rate of cytochrome C on HL-60 cells was detected by MTT, the morphology of HL-60 cells was observed by light microscopy and fluorescence microscopy, the changes of apoptosis rate and cell cycle were assayed by flow cytometry (FCM), DNA ladder was investigated on electrophoresis, the expression changes of bax and bcl-2 mRNA were examined by RT-PCR, when HL-60 cells were treated with different concentrations of cytochrome C for 24 hours. The results showed that the inhibition rate increased with increase of the cytochrome C concentration within 0 - 150 mg/L; when treated with 0 - 37.5 mg/L cytochtome C for 24 hours, the percentage of apoptotic HL-60 cells increased with the dose increasing, and the typical apoptotic cells and the apoptotic DNA ladder were observed. At the same time, within this range of concentration, the expression of bcl-2 mRNA decreased gradually and the expression of bax increased gradually. When the cytochrome C concentration was higher than 37.5 mg/L, the percentage of apoptotic HL-60 cells not increased, but decreased, while the cells necrosed. The above metioned results suggested that at certain range of concentration of cytochrome C, apoptosis or necrosis can be induced by cytochrome C, and cell cycle arrests at G(1) phase in HL-60 cells, the percentage of apoptotic cells and the changes of expression of bax and bcl-2 depend on the dose of cytochrome C. The mechanism that cytochtome C induced apoptosis in HL-60 cells may be related to the activation of bax and inhibition of bcl-2.

Expression of CXCR4 in Acute Leukemic Cells of Children and Its Signific ance / 实用儿科临床杂志

Objective To analyze the chemokine receptor CXCR4 expression in acute leukemic cells of children and its relationship with extramedullary infiltration.Methods The immunotypes of cases of acute leukemia in children and the expression of CXCR4 in marrow leukemic cells were studied by flow cytometry respectively. The relationship between CXCR4 expression and extramedullary infiltration of leukemic cells were analyzed by statistical method.Results The expression rates of CXCR4 in ALL children were higher than those in NALL children(P

Significance of Serum Interleukin-15 Levels in Children with Acute Leukemia / 实用儿科临床杂志

Objective To investigate the levels of interleukin-15(IL-15) in children with acute leukemia and contribute to understand their function on acute leukemia about the process.Methods Every group of acute lymphocytic leukemia(ALL),acute nonlymphocytic leukemia(ANLL) or non-leukemia group had 20 children who did not receive any treatment. Peripheral blood of every group and 20 other healthy children were aspirated .The levels of IL-15 in serum was analyzed by enzyme linked immunosorbent assay (ELISA).Results The levels of IL-15 in ALL group,ANLL group, non-leukemia group and healthy children were (34.37?2.8) ng/L, (29.61?3.2 )ng/L, (117.54?3.9) ng/L, (122.23?4.2) ng/L. Compared with the levels of the healthy group or the non-leukemia group, the difference of ALL or ANLL was signicant in statistics(q=3.95,4.03 Pa0.05).Conclusion Detection of serum IL-15 levels may have clinical significance in judging the anti-tumor immune condition in children with leukemia.

The study of hematopoietic cell reaction to interleukin-15 in children with myelodysplastic syndrome / 实用儿科临床杂志

Objective To investigate children′s myelodysplastic hematopoietic cells reaction to interleukin (IL)-15.Methods CD 34 + cells in bone marrow from 18 myelodysplast syndrome(MDS) patients were purified by an immunomagnetic beads sorting system. Apoptosis of hematopoietic precursors was assayed by propidium iodine staining and flow cytometric analysis.Results On 8th cultured day,when IL-15 concentration was between 0-100 ng/ml,it could suppress apoptosis of hematopoietic cells in MDS patients in a dose-and- time dependent manner. IL-15 in study group significanthy lower than that of control group.Conclusion IL-15 may partly suppress apoptosis of hematopoietic cells in MDS patients.