[Influence of mesenchymal stem cells on proliferation of HL-60].

The objective of this study was to explore the effect of mesenchymal stem cells (MSC) on HL-60 proliferation and its molecular mechanism. HL-60 cells co-cultivated with MSC were used as experiment group, and the cells cultivated solely were taken as control group. HL-60 cells in the two groups were counted at different time. The time-quantity curve was drawn. The cell cycle and apoptosis ratio of HL-60 cells were compared between the two groups and expressions of Survivin and Bcl-2 protein were detected by flow cytometry. The results showed that HL-60 cells cultivated with MSC were obviously inhibited, especially on day 5 and 7 (P < 0.01). HL-60 cells were distributed on the phase of G(0)/G(1) [control group (47.0 ± 9.0)% vs experiment group (70.0 ± 16.0)%, P = 0.003], and apoptotic peak appeared. Both of Survivin and Bcl-2 protein expressions in HL-60 cells decreased [Bcl-2 protein in control group (63.0 ± 9.1)% vs experiment group (50.0 ± 14.1)%, P = 0.045; Survivin in control group (94.0 ± 9.3)% vs experiment group (77.0 ± 11.8)%, P = 0.006]. It is concluded that the MSC can inhibit HL-60 cell proliferation, and promote HL-60 cell apoptosis.

[Influences of bone marrow mesenchymal stem cells in patients with acute myeloid leukemia and non-leukemia on HL-60 cells -- a comparison study].

This study was aimed to compare the influences of bone marrow mesenchymal stem cells (BMMSCs) from patients with acute myeloid leukemia (AML), AML patients with complete remission (CR) and non-leukemia patients on HL-60 cells. The HL-60 cells were divided into three groups: group of co-cultivation with BMMSCs of AML patients, group of co-cultivation with BMMSCs of AML patients with CR and group of co-cultivation with BMMSCs of non-leukemia patients. The count of HL-60 cells, the CD11b and survivin expression of HL-60 cells, the cell cycle distribution of the HL-60 cells in 3 groups were compared by flow cytometry, the morphology and differentiation rate of HL-60 cells in 3 groups were observed and compared by microscopy. The results showed that there were no differences in HL-60 cell count at five and seven days, in HL-60 distribution at the G(0)/G(1) phase, in survivin and CD 11b expressions in 3 groups. All cells of 3 groups began to mature, and the differentiation rates in 3 groups were 18.0 +/- 3, 17.0 +/- 1.3 and 19.0 +/- 2.0 respectively, therefore there were no significant differences between the 3 groups (p = 0.23). It is concluded that there is no influence of BMMSCs in 3 groups on the proliferation and differentiation of HL-60 cells.

[Comparison of biological characteristics of bone marrow mesenchymal stem cells in acute myeloid leukemia with those from non-leukemia].

This study was aimed to compare partial biological characteristics of bone marrow mesenchymal stem cells (BMMSCs) in AML patients, AML patients with complete remission (CR) and non-leukemia patients. The bone marrow (BM) MSCs were divided into 3 groups: group of MSCs from AML patients, group of MSCs from AML patients with CR, group of MSCs from non-leukemia patients. The morphologic features of MSCs were observed by light microscopy; CFU-F numbers of MSCs were counted after Wright-Giemsa staining in situ; the fusion times of MSCs were determined; the growth curves of MSCs were drawn by counting cell numbers; the immunophenotypes and cell cycle of MSCs were detected by flow cytometry; the DI values of MSCs were calculated. The results showed that the morphologic features of MSCs in 3 groups did not display difference; there was significant difference (p < 0.01) of CFU-F numbers in 3 groups, while CFU-F number of MSCs in AML group was minimal; there was significant difference of MSC fusion time in 3 groups, while fusion time of MSCs in AML group was most long; the growth curves of MSCs in 3 groups were similar; MSCs in 3 groups highly expressed CD105 and CD106, but not expressed CD45; the cell distribution ratios at phase of G(0)/G(1) for MSCs in 3 groups were 89.9 +/- 4.0%, 90.2 +/- 3.0% and 91.0 +/- 3.0% respectively; the DI values of MSCs in 3 groups were between 0.9 and 1.1. It is concluded that no significant difference of biological characteristics of the second generations of MSCs is found between those in leukemia and non-leukemia patients.

[Change in coagulation after open abdominal wound subjected to seawater immersion].

OBJECTIVE: To explore the effects of hyperosmotic fluid and low temperature on hemorrhage and coagulation system after immersion of dogs with open abdominal wounds in seawater. METHODS: Twenty healthy dogs were subjected to open abdominal injury, then dogs were randomized equally into two groups: the control group (n=10) (without seawater immersion) and seawater immersion group (n=10). The variables of prothrombin time (PT), partial thromboplastin time (APTT), D-dimer and factor II, granule membrane protein-140 (GMP-140), endothelin-1 (ET-1) were determined. RESULTS: PT and APTT were significantly prolonged. D-dimer, GMP-140, and ET-1 were increased, while factor II was decreased after the dog with open abdominal wound was immersed in seawater (all P<0.05). Compared with the variables of control group, PT, APTT, D-dimer and factor II, ET-1 in seawater immersion group had markedly changed (all P<0.05) except GMP-140 at different time points (all P>0.05). CONCLUSION: Obvious vascular endothelial cell dysfunction, platelet activation, inhibition of coagulation factor activity, coagulopathy, and disorders in thrombosis and fibrinolysis system activation occur after dogs with open abdominal wounds are immersed in seawater.

[Comparison of different cryopreservation systems for peripheral blood stem cells].

AIM: To explore proper cryopreservative systems for hematopoietic stem cells. METHODS: Peripheral blood mononuclear cells from 20 persons were mixed with different cryopreservative agent, dimethyl suflfoxide (DMSO) or combination of DMSO and hydroxyethyl starch (HES), then cooled in -80 degrees C low temperature refrigerator (Refr) or autocontrolled programmed cryogenic system (PCS), preserved in Refr or in liquid nitrogen. GM-CFU, LTC-IC, CD34+ cells and typeran blue resistance (TBR) were assayed after different period of cryopreservation. RESULTS: The recovery rates of CFU-GM, LTC-IC, CD34+ cells and TBR in peripheral blood mononuclear cells which were cooled and preserved in Refr with 5% DMSO-6% HES were 82.2% +/- 14.7%, 83.0% +/- 12.2%, 94.2% +/- 4.3% and 97.7% +/- 3.9% respectively, significantly higher than that in Refr with 10% DMSO (P < 0.05). When cells were cryopreservated with the same cryopreservatives, there was no significantly difference of recovery rate in group of Refr and group of Refr with PCS. Meanwhile, there was not significantly difference of recovery rate among all three groups, preserved in Refr ahead of liquid nitrogen, in Refr merely, in liquid nitrogen with PCS within one year (p > 0.05). However, the recovery rate of CFU-GM, LTC- IC, CD34+ cells and TBR decreased dramatically if cells were cooled and preserved in Refr for two years. After cells were thawed, the cell activity declined gradually at room temperature if the cryopreservatives were not removed or diluted. The cell activity of 10% DMSO group was affected more than that of 5% DMSO-6% HES group. CONCLUSION: 5% DMSO-6% HES is better than 10% DMSO as cryopreservatives for hematopoietic stem cells. Refr cryopreservation is a simple and effective method if cells would be cryopreserved for less than one year. If cells would be cryopreserved for more than one year, liquid nitrogen cryopreservation should be recommended. The cryopreservatives should be diluted or removed immediately after cells were thawed.

Detecting PML-RARalpha transcript in acute promyelocytic leukemia using real-time quantitative RT-PCR.

BACKGROUND: Real-time quantitative RT-PCR (RQ-PCR) assay has become a vital tool to monitor residual disease of leukemia. However, the complexity and standardization of RQ-PCR should never be overlooked and the results should be interpreted cautiously in clinical conditions. We aimed to assess the methodology of RQ-PCR and its clinical applications in monitoring molecular kinetics of 36 newly diagnosed cases of acute promyelocytic leukemia patients with t (15; 17) from October 2004 to December 2005. METHODS: All the TaqMan probe-based RQ-PCR reactions and analysis were performed on an ABI-PRISM 7,500 platform. The quantitation of PML-RARalpha transcripts was represented by the normalized quotient, that is, PML-RARalpha transcript copies divided by ABL transcript copies. According to induction therapy, the patients were classed into two groups: group 1 (n = 23), three-drug combination including arsenics, all-trans retinoic acid and mitoxantrone; and group 2 (n = 13), two-drug combination from all-trans retinoic acid, arsenics and mitoxantrone. RESULTS: The sensitivity of RQ-PCR was 1 per 10(5) cells and 5 copies of the PML-RARalpha transcript could be reproducibly detected. No false positive results occurred in 40 non-acute promyelocytic leukemia samples. Optimal amplification efficiency could be attained, which was determined by the slope of the standard curves (slope: -3.2 - -3.7). The inter-assay and intra-assay variation coefficients of the method were 1.01% and 0.56% respectively. Although the time to attain hematological complete remission was similar in both groups, the time to achieve molecular remission of group 1 was significantly shorter than that of group 2 (61 days vs 75 days, P = 0.034). The rate of molecular remission within 70 days was higher in group 1 than in group 2 (75.00% vs 38.46%, P = 0.036). Compared with pretreatment, median reduction of the PML-RARalpha transcript before first consolidation therapy differed significantly between group 1 and group 2 (log scale, 3.15 vs 2.31, P = 0.024). Interestingly, we found that PML-RARalpha transcript levels temporarily increased in bone marrow (7 patients) and peripheral blood (22 patients) samples of patients during induction therapy in both groups. CONCLUSIONS: The RQ-PCR assay is reliable for the detection of PML-RARalpha transcripts. Arsenics, all-trans retinoic acid and mitoxantrone triad induction treatment of acute promyelocytic leukemia is superior to two-drug combination induction therapy in terms of the molecular response.

[Influence of cryopreservation on leukemic dendritic cells derived from leukemia patients].

This study was aimed to investigate the influence of cryopreservation on biological properties and function of leukemic dendritic cells (L-DCs) derived from patients with acute or chronic leukemia. Some fresh leukemic cells were detected immediately; some were cultured immediately; some were cryopreserved in -80 degrees C with 5% DMSO-6% HES as cryopreservor. After being thawed, they were cultured. The combination of rhGM-CSF, rhIL-4, rhTNF-alpha and other cytokines were added into the culture system. 12 days later, L-DCs were assayed for morphology, immunophenotype, mixed lymphocytic reaction (MLR) and CTL cytotoxicity on autologous leukemic cells. The results showed that both fresh and cryopreserved leukemic cells obtained from patients with acute or chronic leukemia revealed typical DC morphologically by means of using combinations of cytokines in culture, but there was no significant difference between pre-or post cryopreservations. L-DCs also upregulated the expression of CD80, CD54, HLA-DR, CD1a, CD83 and CD86, and downregulated the expression of CD14, but there was also no difference as compared with L-DCs befor cryopreservation. L-DCs derived from leukemic cells were also capable of stimulating MLR and inducing CTL which could kill autologous leukemic cells obviously. It is concluded that leukemic cells, regardless of fresh or frozen, can induce L-DCs after culture with cytokine combination. The L-DCs can induce CTL targeting autologous leukemic cells, and may be used to treat MRD as immunotherapy. The induction and biological properties of L-DCs are not influenced by cryopreservation.

[Effect of total Panax notoginseng saponins on inducing differentiation of mononuclear cells in human cord blood into endothelial cells].

To investigate the effect of total panax notoginseng saponins (tPNS) to induce the differentiation of mononuclear cells (MNC) in cord blood into endothelial cells, the DMEM culture media containing tPNS were used to induce the MNC of cord blood. Then, the morphology of the adherent cells was observed by the light microscopy and the fluorescence microscopy, the changes of cell surface markers (UEA-1), function marker (vWF) and CD31 were detected by flow cytometry. The results showed that the number of adherent cells produced by 250 mg/L tPNS and the positive rate of cells expressing CD31 and UEA-1 were higher than those in the groups of other concentrations (P < 0.05). There was no significant difference in the number of adherent cells expressing CD31 and UEA-1 between 50 ng/ml VEGF + 250 mg/L tPNS and 50 ng/ml VEGF. It is concluded that the traditional Chinese drug tPNS can induce partial MNC in the cord blood to differentiate into endothelial cells. No synergistic effect has been found between tPNS and VEGF.

[Vascular endothelial progenitor cells and cytokines related to hematopoietic regulation--review].

Endothelial progenitor cell (EPC) is a subtype of progenitor cells that can circulate, proliferate and have the ability to differentiate into mature endothelial cells. Exploration of EPC have significance to understand the vascular forming process of adults physiologically and pathologically. Cytokines, especially haematopoiesis-related cytokines, cellular stimulating factors in the body also influence EPC. In this review, the advances of research on the relationship between EPC and some cytokines related with haematopoiesis (G-CSF, EPO, HAPO and IL-18) and the potential signal transduction pathway were summarised.

[Inhibitive effect of previously activated psoralens on K562 cell proliferation].

The objective was to observe the influence of previously activated psoralens on the proliferation of K562 cells, and to provide laboratory data for its clinical usage. K562 cells were treated separately with previously and late activated psoralens, then their trypan blue exclusion inhibited rates (TBIR), cell proliferation inhibited rates (CPIR) and colony forming inhibited rates (CFIR) after culture were compared. The results showed that previously activated psoralens displayed an inhibiting effect on the proliferation of K562 cells with a dose-effect relationship. There was no obvious difference between previously and late activated psoralens on TBIR, CPIR and CFIR. In order to exert the inhibitive effect of previously activated psoralens, the time of ultraviolet ray exposure should be 10 minutes at least, and longer than 12 hours for inhibiting K562. The inhibitive effect of previously activated psoratens decreased as the time interval from activation to its use was prolonged. The inhibiting effect of previously activated psoralens was strongest within 6 hours after activation. In conclusion, both previously and late activated psoralens show inhibiting effects on the proliferation of K562, which may be able to use an antineoplastic drug in clinic.

[Enhanced apoptosis-inducing effect of etoposide on leukemic cell lines M-07e and TF-1 by the proteasome inhibitor Z-LLL-CHO].

Recent researches indicate that ubiquitin-protea some pathway plays an important role in apoptosis regulation. Proteasome inhibitors induce apoptosis in many kinds of neoplastic cells, thus provide a great opportunity for exploring synergy of proteasome inhibitors and other apoptosis-inducing agents. In this study, the effect of the proteasome inhibitor Z-LLL-CHO combined with etoposide (VP16) on leukemic cell lines M-07e and TF-1 was investigated by MTT assay, trypan blue exclusion, flow cytometry and Western blot. The results showed that the combination of Z-LLL-CHO and VP16 was much more effective than either agents alone in promoting cytotoxicity in both cell lines evaluated. Accumulation of cells in S + G2/M phase of the cell cycle was observed in the cells treated with VP16 and Z-LLL-CHO alone, while apparent increase of sub-G0/G1 fraction was detected in cells treated with combination of the agents. The cleavage of Bcl-2 into a shortened 22 kD fragment was detected in M-07e cells exposed to either agents alone, and the fraction of 22 kD fragment was increased in the cells treated with combination of the agents. In conclusion, the combination of Z-LLL-CHO and VP16 enhanced their individual cytotoxic effect by inducing apoptosis, in which increase of S + G2/M fraction in cell cycle as well as the enhanced cleavage of Bcl-2 are the possible mechanism of the additive effect on leukemic cells by Z-LLL-CHO and VP16.

[Induction of Apoptosis in Leukemic Cells by Inhibiting the Ubiquitin-Proteasome Pathway and Its Possible Mechanism]

The ubiquitin-proteasome pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. Recently, proteasome inhibitors have been shown to induce apoptosis in many kinds of human malignant cells. In this study, the mechanism of apoptosis induced by proteasome inhibitor in leukemic cells was examined. Evaluated by MTT assay, treatment of leukemic cells with Z-LLL-CHO, a reversible proteasome inhibitor, induced cell death in a dose-dependent manner. Appearance of the sub G(0)/G(1) fraction of cell cycle observed in flow cytometry assay suggested the induction of apoptosis, which was further proved by typical DNA ladder and morphological study. Western blot displayed the cleavage of bcl-2 into a shortened 22 kD fragment and the decrease in the levels of caspase-3 precursor. A highly sensitive colorimetric assay was employed and the elevation of caspase-3 activity was detected in both cell lines after treatment with Z-LLL-CHO. By comparison, these results showed that the leukemic cell line M-07e and KG-1a, which both express bcl-2 at a relative high level, had different susceptibility to undergo apoptosis induced by Z-LLL-CHO, which possibly due to their different levels of expression and activation of caspase-3 precursor, as well as their different degree of bcl-2 cleavage after treated by Z-LLL-CHO.