Effect of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells on the Proliferation and Apoptosis of Leukemic Cells and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of human umbilical cord blood-derived mesenchymal stem cells(HUCMSC) on the leukemic cell line HL-60 and acute lymphoblastic leukemia cell line Jurkat as well as the role of CXCL12/CXCR4.</p><p><b>METHODS</b>HL-60 cells and Jurkat cells were co-cultured with human umbilical cord blood mesenchymal stem cell (HUCMSC), and the model was treated with G-CSF, AMD3100 and their combination. The cell viability and cell cycle were measured by Cell Counting Kit-8 (CCK-8), the apoptosis and the cell-cycle analysis were assessed by flow cytometry with the Annexin V/PI double staining. The expression of surface CXCR4 protein and total CXCR4 protein of leukemic cells were detected by flow cytometry and Western blot respectively.</p><p><b>RESULTS</b>HUCMSC could decrease the viability of HL-60 cells and Jurkat cells, as well as the percentage of apoptotic cells, they could also increase the number of G/Gcells, while G-CSF and AMD3100 could reduce the proliferation of HL-60 cells and Jurkat cells in HUCMSC co-culture model, destructed the anti-apoptotic effect of HUCMSC on HL-60 cells and Jurkat cells, and the combination of 2 drugs resulted in a synergistic effect. The G-CSF could reduce the expression of surface CXCR4 protein and total CXCR4 protein in leukemic cells, while AMD3100 could only decrease the expression of surface CXCR4 protein of leukemia cell membrane, having no effect on the expression of CXCR4 protein in cytoplasm.</p><p><b>CONCLUSION</b>Human umbilical cord blood mesenchymal stem cells can inhibit the proliferation and apoptosis of acute leukemia cells and increase the number of G/Gphase cells in leukemic cells. The AMD3100 can decrease the expression of surface CXCR4 protein in leukemia cells, G-CSF can decrease expression of total CXCR4 protein as well as membrane CXCR4 protein. Both of them can block the CXCL12/CXCR4 signal axis, weakening the relationship between leukemia cells and microenvironment. And on the basic of HUCMSC influenced leukemia cells' growth and proliferation, the cell viability will be weakened, its apoptosis will be promoted, and the percentage of G/Gphase cells in leukemia cells will be decreased.</p>

Expression of HOXB4, PRDM16 and HOXA9 in Patients with Acute Myeloid Leukemia and Its Clinical Significance / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate HOXB4, PRDM16 and HOXA9 gene expression in patients with acute myeloid leukemia (AML) and its clinical significance.</p><p><b>METHODS</b>Real-time quantitative PCR (RT-qPCR) with SYBR Green assay was used to detect the expression of HOXB4, PRDM16 and HOXA9 gene in AML patients (40 cases), the patients with complete remission (9 cases) and patients with non-malignant hematologic diseases as control (10 cases). The relationship between the expression levels of gene HOXB4, PRDM16, HOXA9 and clinical features was investigated by statistical analysis.</p><p><b>RESULTS</b>The gene expression levels of HOXB4, PRDM16, HOXA9 in newly diagnosed or relapsed AML patients were significantly higher than those in patients with non-malignant hematologic disease (P < 0.05). It was observed that the expression of HOXB4 gene in newly diagnosed or relapsed patients positively correlates with leukemic blasts in bone marrow (r = 0.39). The expression levels of HOXB4, PRDM16 and HOXA9 positively correlate with each other. There was statistical significance among gene expressions in different phases (newly diagnosed, relapse, remission). No correlation was observed between expression levels of HOXB4, PRDM16, HOXA9 and chromosome risk status. It was noticed that expression levels of HOXB4, PRDM16, HOXA9 genes were lower in the patients achieved remission after two courses of chemotherapy than those in the other. And high expression group of each gene had a lower remission rate than that in the low expression group.</p><p><b>CONCLUSION</b>The expression level of HOXB4, PRDM16, HOXA9 genes and leukemic blasts somewhat correlate with curative effect and prognosis. The expression of HOXB4, PRDM16, HOXA9 genes is higher in newly diagnosed and relapsed leukemia patients, and lower in the patients acquired CR/PR. High expression of HOXB4, PRDM16, HOXA9 genes predicts an adverse prognosis.</p>

Effect of 5-aza-2'-deoxycytidine combined with trichostatin A on RPMI-8226 cell proliferation, apoptosis and DLC-1 gene expression / 中国实验血液学杂志

This study was aimed to investigate the effects of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) and histone deacetylase inhibitor trichostatin A (TSA) on DLC-1 gene transcription regulation and molecular biological behaviours in the human multiple myeloma RPMI-8226 cells. The cells were treated respectively with 5-Aza-CdR and TSA alone, or the both combination; the cell proliferation and apoptosis, DLC-1 expression, the protein expression of Ras homolog family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac1) were examined by CCK-8 method, RT-PCR and ELISA, respectively. The results showed that the 5-Aza-CdR and TSA had cell growth inhibitory and apoptosis-inducing effects in dose-dependent manner (P < 0.05). Compared with a single drug (5-Aza-CdR or TSA alone), the effects were significantly enhanced after treatment with the combination of 5-Aza-CdR and TSA (P < 0.05). DLC-1 was weakly expressed in the control group; the treatment with 5-Aza-CdR alone enhanced its re-expression dose-dependently (P < 0.05). Compared with 5-Aza-CdR alone, 5-Aza-CdR plus TSA enhanced DLC-1 re-expression significantly.Compared with the control, 5-Aza-CdR and TSA significantly decreased RhoA and Rac1 protein expression (P < 0.05). It is concluded that 5-Aza-CdR and TSA can effectively reverse DLC-1 expression of RPMI-8226 cells; TSA has a synergistic effect on its re-expression. 5-Aza-CdR and TSA have significant cell growth inhibitory and apoptosis-inducing effects on RPMI-8226 cells. These effects may be related to the inhibition of Rho/Rho kinase signalling pathway.

Recombinant human thrombopoietin in combination with cyclosporin A as a novel therapy in corticosteroid-resistant primary immune thrombocytopenia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The management of patients with refractory immune thrombocytopenia (ITP) is challenging, as there is no standard treatment option. The aim of this study was to investigate the efficacy of recombinant human thrombopoietin (rhTPO) in combination with cyclosporin A (CsA) for the management of patients with corticosteroid-resistant primary ITP.</p><p><b>METHODS</b>Thirty-six patients with corticosteroid-resistant ITP were randomly divided into an observation group and control group. In the observation group, 19 patients received subcutaneous injection of rhTPO at a dose of 1 µg/kg (300 U/kg) once daily up to day 14. Simultaneously they also received oral CsA at a dose of 1.5-2.0 mg/kg twice daily for three months. In the control group, rhTPO alone was administered subcutaneously at 1 µg/kg once daily in the other 17 ITP patients for 14 consecutive days and then the treatment was withdrawn.</p><p><b>RESULTS</b>There was no significant difference in the response rate at the end of the first week after treatment initiation between the observation group and the control group (63.2% vs. 58.8%, P > 0.05), neither was there at the end of the second week (89.5% vs. 94.1%, P > 0.05). However, the relapse rate in the observation group was significantly lower than that in control group at the end of the first (17.7% vs. 50.0%, P < 0.05), second (29.4% vs. 68.8%, P < 0.05) and the third month (29.4% vs. 87.5%, P < 0.01). In addition, rhTPO plus CsA were well tolerated and adverse events recorded were mild.</p><p><b>CONCLUSIONS</b>Combination therapy with rhTPO and CsA was effective in the management of patients with corticosteroidresistant ITP, with a relatively short time to response and low recurrence rate. It might be considered as a potential secondline treatment regimen for ITP.</p>

Effects of HOXB4-transfecting directly and HOXB4-transfected HUCMSC on in vitro expansion of human umbilical cord blood CD34+ cells / 中国实验血液学杂志

This study was purposed to investigate the difference of nucleated cell (NC) count, CD34(+) cell ratio and expansion multiple, cell cycle and colony formation capability in in vitro expanded human umbilical cord blood CD34(+) cells from HOXB4-transfecting directly and HOXB4-transfected human umbilical cord mesenchymal stem cells (HUCMSC) by means of prepared feeder layers of HUCMSC. The HUCMSC were divided into 2 groups:first group, in which HOXB4 gene was transfected into HUCMSC by using lentiviral vecfor, and feeder layers were set up; and second group in which feeder layers for HUCMSC of non-transfected HOXB4 gene were set up. The CD34(+) cells were separated from HUCB by magmatic activated cell sorting(MACS). After culture in medium with cytokines for 2 days, CD34(+) cells were divided into 5 groups, including control group and experimental group. The control groups included CD34(+) cells as group A (blank control group) and GFP-CD34(+) cells as group B (negative control group) and experimental groups included HOXB4-CD34(+) cells as group C, HUCMSC+CD34(+) cells as group D, HOXB4-HUCMSC+ CD34(+) cells as group E and cells in all groups were cultured in vitro. The number of nucleated cells were counted at day 6, 10, 14 of culture and CD34 immunophenotypes, cell cycle and colony forming capability were measured at day 10 of culture in different conditions. The results indicated that HOXB4 gene could be transfected into HUCMSC by lentiviral vector and feeder layers were set up successfully. After culture for 14 days, the nucleated cells in 5 groups could be amplified effectively, and the expansion levels in 5 groups were in order HOXB4-HUCMSC+CD34(+) cell group> HOXB4-CD34(+) cell group>HUCMSC+CD34(+) cell group> control groups (P < 0.05). At day 10 of in vitro expansion the CD34(+) cell percentage decreased significantly in all groups, while the number of CD34(+) cell increased in experiment groups, which were in order HOXB4-CD34(+) cells group> HOXB4-HUCMSC+CD34(+) cell group>HUCMSC+CD34(+) cell group>control groups (P < 0.05). The cell cycle detection showed that the percentage of cells in S+G2/M phase in experiment groups were higher than that in control groups (P < 0.05), and percentage of cells in HOXB4-HUCMSC+CD34(+) cells group was higher (41.57%) than that in HOXB4-CD34(+) cells group(37.87%) and HUCMSC+CD34(+) cell group (28.65%) (P < 0.05). There was no statistical difference in the CFU number between HOXB4-HUCMSC+CD34(+) cell group and HOXB4-CD34(+) cell group, which were both higher than that in HUCMSC+CD34(+) cell group and control groups (P < 0.05).It is concluded that the CD34(+) cells cultured on HOXB4-HUCMSC feeder layers can be amplified significantly and kept the characteristics of stem cells, The feeder lager of HOXB4-HUCMSC is relative safe for amplification of CD34(+) cells in vitro, it possesses the potential useful value.

Construction of lentivirus vector containing human homeobox gene HOXB4 and its expression in human umbilical cord mesenchymal stem cells / 中国实验血液学杂志

This study was purposed to construct lentivirus vector containing human homeobox gene HOXB4 and explore changes of human umbilical cord mesenchymal stem cells (HUCMSC) after infected with HOXB4 mediated by lentivirus. PCR amplification was performed to obtain HOXB4, which was cloned in lenti-shuttle vector. Four-plasmid lentivirus packaging system was used to transfect HEK293T cells. After 48 h, lentivirus Lenti-HOXB4 was harvested and lentivirus titer was determined. Lenti-HOXB4 was used to infect HUCMSC. The infected cells were observed under inverted fluorescence microscope to determine the optimal multiplicity of infection (MOI). Meanwhile, RT-PCR, immune fluorescence staining, CCK-8 and flow cytometry (FCM) were used to determine the expression of HOXB4 and its effect on cell growth. The results indicated that lenti-HOXB4 was successfully obtained by co-transfecting the 293T cells with four plasmids. The determined virus titer was 3×10(8) TU/ml; when MOI was 20. Lenti-HOXB4 had a high transfection rate in HUCMSC, over 80%. In HUCMSC infected with lenti-HOXB4, the expression of target gene could be detected both at mRNA and protein levels. It could promote the proliferation of HUCMSC. FCM results indicated HOXB4 gene did not significantly influence the surface marker of HUCMSC. It is concluded that HOXB4 gene can promote the high proliferation of HUCMSC and does not significantly influence the expression of the surface marker of HUCMSC.

Construction and identification of a eukaryotic expression vector containing human homeobox gene / 中国实验血液学杂志

In order to investigate the special role of HOXB4 in expansion and self renewal of hematopoietic stem cells, the cDNA of HOXB4 was extracted and cloned from umbilical cord blood mononuclear cells by using RT-PCR. Then the eukaryotic expression bicistronic plasmid vector pIRES2-EGFP/HOXB4 was designed and constructed after cutting HOXB4 and pIRES2-EGFP respectively by restriction enzyme EcoRI and BamHI. The recombinant plasmid was delivered into competent cells of Escherichia coli. The successful construction of plasmid was confirmed by the identification of endonuclease cutting and sequencing. The results showed that the HOXB4 cDNA was cloned successfully from umbilical cord blood mononuclear cells and the recombinant eukaryotic expression bicistronic plasmid vector was constructed, and then introduced it into 293T cells successfully. It is concluded that a pIRES2-EGFP/HoxB1 eukaryotic expression bicistronic plasmid vector has been constructed successfully, which results provide a useful material basis for exploration of HoxB4 function in the proliferation and differentiation of hematopoietic cells.

Effect of dendritic cells on expansion and function of autologous natural killer cells in vitro / 中国实验血液学杂志

The objective of this study was to investigate the effect of dendritic cells (DCs) on expansion and function of autologous natural killer (NK) cells and its mechanism in vitro. NK cells were expanded from peripheral blood mononuclear cells (PBMNCs) of healthy volunteers in stem cell growth medium (SCGM) supplemented with rhIL-2 (control group) in 24-well culture plates at 37 degrees C in a humidified CO(2)-containing atmosphere. NK cells were cultured with autologous DCs in the ratio of 5 to 1 (group 5:1) or 1 to 1 (group 1:1) from day 10 after expansion. Total cells of every group were counted and the expression of CD3, CD16/56 on the surface of NK cells was assayed by flow cytometry on days 7, 14 and 21 to calculate the expansion of NK cells. Cytotoxicity of expanded NK cells against K562 cells was assayed by MTT method. TNF-alpha and IL-12p70 were detected in culture supernatants by sandwich ELISA. The results indicated that the expansion and cytotoxicity of NK cells were improved after mixed with autologous DCs. Furthermore, when DCs were mixed with NK cells, the ratio of DCs to NK cells was higher, the expansion and cytotoxicity NK cells were higher. On day 14, the expansion multiple in control, group 5:1 and group 1:1 were 16.26 +/- 1.58, 29.25 +/- 4.01 and 21.23 +/- 2.91 respectively. The expansion multiple of group 5:1 was much higher than that of the other two groups (p < 0.05). The expressions of CD3(-), CD56/16(+) on surface of NK cells in control, group 5:1, group 1:1 were (34.8 +/- 5.1)%, (64.6 +/- 7.8)% and (50.6 +/- 8.7)% respectively and that of group 5:1 was the highest (p < 0.05). The cytotoxicities against K562 cells in control, group 5:1 and group 1:1 were (63.7 +/- 3.8)%, (87.4 +/- 6.8)% and (75.4 +/- 6.3)% respectively. The cytotoxicity of group 5:1 was higher than that in the other two groups also (p < 0.05). TNF-alpha and IL-12p70 levels in culture supernatants when DCs and NK cells were mixed in the ratio of 5 to 1 were much higher than those in culture supernatants of DCs and NK cells alone or in culture supernatants when DCs and NK cells were mixed in the ratio of 1 to 1 (p < 0.05). It is concluded that the expansion and cytotoxicity of NK cells can be improved by DCs and it depended on the mixed ratio of DCs to NK cells. The elevated expansion of NK cells by DCs bears relation to IL-12 produced by DCs. The enhanced cytotoxicity of NK cells is associated with TNF-alpha secreted by NK cells.

Study on rapid generation of dendritic cells from K562 cell line induced by A23187 alone / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore a simple, rapid and efficient way to generate dendritic cells from leukemic cells.</p><p><b>METHODS</b>K562 cells were cultured with calcium ionosphere A23187 alone, A23187 plus GM-CSF, or a DC differentiation cocktail consisting of GM-CSF, IL-4 and TNF-alpha, respectively. The expression of surface markers of induced DCs was analyzed by flow cytometry. The K562-DCs stimulating the proliferation of allo-genetic naive T cells and inducing cytotoxicity of T cells were determined by MTT assay.</p><p><b>RESULTS</b>Microscopic examination revealed that under all the three culture conditions, K562 cells became displaying DC morphology. At 72 hours in the two culture systems containing A23187, there were higher proportions of cells with dendritic morphology [(69.5 +/- 17.2)% and (73.1 +/- 13.9)%, respectively] than that in the cocktail system [(28.5 +/- 12.3)%] (P < 0.05). And the same did when cultured for 7 days [(69.5 +/- 17.2)%, (73.1 +/- 13.9)% respectively vs (51.2 +/- 10.7)%, P < 0.05]. In the 7-day cultures, the percentage of CD1a expressing cells was lower [(8.2 +/- 2.3)% and (10.3 +/- 5.1)% vs (17.2 +/- 1.6)%, respectively] while the CD83 expressing cells was higher [(85.6 +/- 8.8)% and (82.4 +/- 9.1)% vs (77.4 +/- 12.9)%, respectively] compared with that in the cocktail system (P < 0.05). No significant difference was found in the allogeneic T cell proliferation response and induced T cell cytotoxicity between A23187 containing and cocktail groups (P > 0.05).</p><p><b>CONCLUSIONS</b>A23187 treatment is a simple, rapid and efficient in vitro strategy for inducing dendritic cell from leukemic cells.</p>

The induction and function study on dendritic cells derived from blasts from patients with acute myelogenous leukemia / 中国实验血液学杂志

To investigate the induction method and function of dendritic cells (DC) derived from acute myelogenous leukemia (AML) blasts in vitro, cytokine-supplemented suspension cultures of leukemia blasts in 25 AML patients were performed. Mononuclear cells were cultured for 8 to 12 days using recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), recombinant human interleukin-4 (rhIL-4) and recombinant human tumor necrosis factor-alpha (rhTNF-alpha). Morphology, phenotype, cytogenetics, and function of induced cells were studied. The results showed that after culture for 3 days, cells in 20/25 AML cases demonstrated an increase in size with dendritic morphology. After culture for 8 - 9 days, the percentage of such cells reached peak. When cultured for 12 days, the total number of cells and the number of cells with DC morphology decreased greatly. Phenotypic analyses of cells (11/20 cases) were measured by flow cytometry before and after culture. Before culture, cells did not express CD1a, CD80 and CD83, while expressed CD54, CD86 and HLA-DR with low frequency. After culture, cells upregulated CD1a, CD80, CD83, CD54, CD86 and HLA-DR significantly. A marked increase of the T-cell stimulatory capacity could be generated in Allo-MLRs. FISH confirmed the leukemic origin of generated cells. In conclusion, leukemia-derived DC can be generated from AML blasts using cytokine combination (GM-CSF, IL-4, and TNF-alpha) in vitro.

Study on cryopreservative methods for dendritic cells derived from K562 cell line / 中国实验血液学杂志

To investigate the biological properties of cryopreserved dendritic cell (DC) derived from K562 cell line, thus to provide a simple, quick and efficient preservative method of DC for infusion of DC to patients with leukemia after complete remission, fresh DC induced from K562 cell line (K562-DC) was frozen in -196 degrees C liquid nitrogen and -80 degrees C mechanical freezer by method of steps (RPMI 1640 with 10% DMSO, 20% FCS as cryopreservatives), and thawed in different time, respectively. Survivals of cryopreserved and fresh K562-DC, expression of surface antigens, stimulating index (SI) and cytotoxic eliminating rate were detected. The results of fresh induced cells were compared with that of cryopreserved ones. The results showed that before and after frozen in liquid nitrogen, the morphological characteristics of K562-DC had no distinct change; and both their expression rates of surface molecular and capacity to stimulate allogeneic lymphocyte had no statistic significance (P > 0.05). In addition, there were no differences in terms of viability, stimulatory capacity and cytotoxicity of K562-DC from two ways for less than one month (P > 0.05), but there were differences when frozen for more than one month (P < 0.05). It is concluded that there is no significant difference when frozen less than one month between liquid nitrogen and -80 degrees C freezer; but when time is more than one month, K562-DC frozen in -196 degrees C liquid nitrogen is better than that in -80 degrees C freezer.