Showing papers in "Journal of Experimental Hematology in 2006"

Effects of plumbagin on the human acute promyelocytic leukemia cells in vitro

Abstract: According to previous clinical experiences of the authors, plumbago zeylanica was effective against acute promyelocytic leukemia (APL). However, its effectiveness has never been proven experimentally or unequivocally clinically. This study was aimed to investigate the effects of plumbagin on the proliferation, cell cycle and apoptosis of APL cell line NB4 Cells. Cell inhibitory rates were detected by MTT colorimetric assay; morphologic changes were observed under light microscope and transmission electron microscope; apoptosis-inducing effects were determined by DNA gel electrophoresis, annexin V/PI double-stained and PI single-stained flow cytometry. The results demonstrated that 2-15 micromol/L plumbagin inhibited the proliferation of NB4 cells in a dose-dependent manner. The morphologic changes of cell apoptosis, such as chromsome condensation and apoptotic body formation, were observed by light microscope and transmission electron microscope. Cell cycle analysis showed that NB4 cells were blocked in G2/M phase of cell cycle. And plumbagin induced annexin V+/PI- cell increase and DNA fragmentation. There was a correlation between cell apoptosis rates and the concentrations of plumbagin in dose-dependent manner (P < 0.05). It is concluded that for the first time the present study shows that plumbagin can inhibit cell proliferation, block cell cycle and induce apoptosis of APL cell line NB4 cells.

[Thapsigargin-induced apoptosis of K562 cells and its mechanism].

Abstract: The aim was to study the apoptotic induction effect of thapsigargin on leukemia cell line K562 and its possible mechanism. After the treatment of leukemia cell line K562 by thapsigargin, morphological change of apoptotic cells was investigated by AO/EB fluorescent staining under fluorescent microscope; apoptosis rate was determined with annexin V-FITC/PI double staining by flow cytometry; intracellular calcium concentrations ([Ca(2+)]i) were measured by fluorescence spectrophotometer with calcium sensitive fluorescence indicator Fura-2/AM; mitochondrial transmembrance potentials (Delta Psi m) was detected on flow cytometry through staining of Rhodamine (Rh123); the changes of caspase-3, -7, -9, -12, cytochrome C, GRP78 proteins were detected by Western blot. The results showed that K562 cells cultured in 4 micromol/L thapsigargin for 48 hours exhibited typical morphological changes of apoptotic cells under fluorescent microscope, including shrinkage of cell, condensation of chromatin, breakage of nuclear, formation of apoptotic bodies, fluorescence of yellow green and pellet observed in early apoptoyic cells and hyacinth fluorescence of chromatin showed in late apoptotic cells. 24 and 48 hours after exposure to 1, 2, 4, 8 micromol/L thapsigargin, the apoptotic rates of K562 were respectively 7.51%, 11.65%, 23.22%, 30.56% and 12.85%, 20.27%, 31.51%, 44.16%, in dose-dependent manner, and were statistically significant when compared with the controls (P < 0.05). The apoptotic rate of K562 was dose- and time-dependent in experiment range. The enhancement of [Ca(2+)]i and the decrease of the Delta Psi m in K562 cells were induced by thapsigargin and were dose-dependent in experiment range, compared with control, P < 0.05. Western blot results indicated that cleavage and activation of caspase-3, -7, -9, -12, releasing of cytochrome C from mitochondria, upregulation of GRP78 expression at the endoplasmic reticulum were induced in K562 cells after 24 hours exposure of 4 micromol/L thapsigargin. It is concluded that thapsigargin induces endoplasmic reticulum stress-induced apoptosis in K562 cells. Endoplasmic reticulum is a novel important initiatory site of apoptosis in cells; the cleavage and activation of caspase-3, -7, -9, -12 play very important role in endoplasmic reticulum stress-induced apoptosis of K562 cells and is one of the important mechanisms for thapsigargin-induced apoptosis. Thapsigargin-induced apoptosis in K562 cells is associated closely with the disruption of the Delta Psi m and the release of cytochrome C from mitochondria, mitochondria participates in endoplasmic reticulum stress-induced apoptosis in K562 cells.

An iron regulator hepcidin is affected by EPO

Abstract: To investigate the effect of EPO on hepcidin mRNA expression in normal mice, acute inflammatory mice and ACD mice, the acute phase model and ACD model of mouse was produced by subcutaneous injection of turpentine oil. Hepatic hepcidin mRNA expression levels of normal mice, acute inflammatory mice and ACD mice were detected by semi-quantitative retro-transcriptase polymerase chain reaction (RT-PCR) after intra-peritoneal injection of EPO. The results showed that prolonged chronic inflammation did not significantly increase mouse hepatic hepcidin mRNA expression, but a single injection of turpentine oil increased mouse hepatic hepcidin mRNA expression transiently. Intra-peritoneal injection of EPO down-regulated hepatic hepcidin mRNA expression in normal mice, ACD mice and acute inflammatory mice. Hb levels had a negative correlation with hepatic hepcidin mRNA expression levels in ACD mice treated with EPO. It is concluded that the ACD model can be produced by repeated subcutaneous injection of turpentine oil. Hepatic hepcidin mRNA expression increased during the early period of ACD process and then fall to normal level. EPO down-regulate hepcidin mRNA expression under the normal, acute inflammation and chronic inflammation conditions.

Effect of ginseng saponin, arsenic trioxide, beta-elemene combined with CTX on telomere-telomerase system in K562 cell line.

Abstract: This study was aimed to investigate the modulating effects on telomere length and telomerase activity in K562 cells treated by arsenic trioxide, ginseng saponin, beta-elemene alone or in combination with cyclophosphamide (CTX) and to explore the possible mechanism and new therapy for acute leukemia. Human erythroleukemic cell line K562 was co-cultured with the above-mentioned drugs. Cells were collected after 24, 48 and 72 hours for further detection. Telomere length and telomerase activity were detected by Southern-blot and PCR-ELISA respectively. The effects of these drugs were observed at different concentrations and exposure time. The results showed that (1) ginseng saponin, arsenic trioxide, beta-elemene, or CTX could completely inhibit the telomerase activity of K562 cells at proper concentrations and exposure time. The inhibiting effects were enhanced when the three former drugs were used with CTX. Telomerase activity decreased proportionally with the concentrations and length of time. (2) viability of K562 cells was decreased after being co-cultured with arsenic trioxide, ginseng saponin, beta-elemene and CTX. The level of inhibition depends on the concentration and exposure time. (3) telomere length of K562 cells was 5.36 +/- 0.18 kb. After being co-cultured with those drugs for 72 hours, telomere length was 5.90 kb -6.50 kb, significantly longer than that of control (5.18 - 5.35 kb). It is concluded that arsenic trioxide, ginseng saponin, and beta-elemene can inhibit the growth and telomerase activity of K562 cells. The inhibiting effects were enhanced when they were used in combination with CTX. The depression of telomerase activity may be one of the mechanisms of anti-tumor effect. Less dosage and shorter course can be expected when arsenic trioxide, ginseng saponin, and beta-elemene are used in combination with CTX. When telomerase activity was depressed, the telomere length prolonged a little, indicating K562 cell line may extend telomeres by some alternative way other than telomerase activation.

[A new method for 5, 10-methylenetetrahydrofolate reductase single nucleotide polymorphisms genotyping used to study susceptibility of hematological malignancy].

Abstract: The aim of this study was to set up a new method for 5, 10-Methylenetetrahydrofolate reductase (MTHFR) single nucleotide polymorphisms (SNP) genotyping, and to investigate the hereditary susceptibility of hematological malignancy. Prepared an aimed gene microarray based on cDNA microarray theory, dual-color fluorescence hybridization was used to detect SNP loci, and DNA sequencing was performed to confirm the results. The MTHFR C677T SNP loci of 157 controls and 127 patients with hematological malignancies (30 multiple myeloma, 28 non-Hodgkin's lymphoma, 22 acute lymphoblastic leukemia, 40 acute myeloid leukemia, 7 chronic myeloid leukemia) from Jiangsu province were detected. The results showed that after overlapping, homozygous wild type, heterozygote type and homozygous mutant type yielded green, yellow and red fluorescence, respectively. DNA sequencing validated these results. The allele frequency of 677C and 677T in patients and controls were 58.7% and 66.9%, 41.3% and 33.1% respectively, showing statistically significant difference (chi2 = 4.077, P = 0.043). 677TT genotype showed a significantly higher risk of MM (OR = 4.21; 95% CI = 1.50 - 11.83; P = 0.006). It is concluded that this microarray-based method is accurate, high-throughput and inexpensive, suitable for SNP genotyping in a large number of individuals. C677T polymorphisms influence the risk of hematological malignancies. 677TT genotype is susceptive to MM.

[Osteoblasts differentiated from human marrow bone mesenchymal stem cells support hematopoietic stem/progenitor cells from umbilical cord blood].

Abstract: This study was aimed to construct a two-dimensional culture system by using osteoblasts induced from human marrow mesenchymal stem cells (MSC) and to investigate its support effect on survival of hematopoietic stem/progenitor cells for umbilical cord blood (UCB) ex vivo. MSCs were isolated from adult human bone marrow and were cultured, the second generation of MSCs were induced into osteoblasts which were irradiated with 20 Gy gamma rays in a Cobalt 60 source and confluenced into a feeder layer. CD34(+) cells were selected from fresh umbilical cord blood samples by using Microbead Kit of MiniMACS and seeded into the two-dimensional culture system to culture ex vivo without exogenous cytokines. By using colony-forming assay, high proliferative potential colony-forming cell assay, and long-term culture initiating cell assay, the ability of the two-dimensional system to culture HSCs/HPCs was observed. The results showed that the osteoblasts induced from bone marrow MSC in constructed two-dimensional culture system displayed more significant support effect on survival of hematopoietic stem/progenitor cells from umbilical cord blood (UCB) ex vivo, compared with other culture systems, especially on long term HSCs survival ex vivo. It is concluded that the two-dimensional culture system constituted by osteoblasts induced from human MSCs has certain ability of supporting maintenance and multipotency of HSCs/HPCs from umbilical cord blood in vitro, especially sustaining survival of HSC in long-term culture. It has also been proved that osteoblasts play a crucial role in regulation of HSC growth.

[Proliferation inhibition and apoptosis induction of K562 cells by D-limonene].

Abstract: This study was aimed to investigate the effect of D-limonene on K562 leukemia cells and its mechanism. Inhibitory effect of D-limonene on proliferation of K562 leukemia cells was assayed by MTT method and cell apoptosis was detected by flow cytometry and DNA agarose gel electrophoresis, the morphologic change of K562 cells was observed by microscopy. The results showed that when K562 cells were treated with 0.125 - 1.0 mmol/L of D-limonene for 48 hours, the proliferation of K562 cells was obviously inhibited in dose-dependent manner. Typical morphological changes and the typical DNA ladder on agarose gel electrophoresis for analysis of cellular apoptosis were significantly appeared in D-limonene treated K562 cells. Simultaneously, the sub-G1 peak was found in FCM analysis. It is concluded that the D-limonene can inhibit proliferation of K562 cells in dose-dependent manner, cause cell detained at G1 phase and induce apoptosis of K562 cells.

[CD4+ CD25high regulatory T cells in peripheral blood of patients with B cell non-Hodgkin's lymphoma].

Abstract: This study was aimed to analyze the proportion of T cell subsets, CD4(+) CD25(high) regulating T cells (Tr) in peripheral blood of B-NHL patients and their change regularity, and to investigate the immunosuppression mechanism and influence of chemotherapy on immunosuppression function of B-NHL patients. The peripheral blood was collected from 42 patients with B-NHL, 36 patients with B-NHL who achieved partial remission (PR) or complete remission (CR) after 4 - 6 cycles of chemotherapy and 15 healthy controls. By using monoclonal antibodies, the blood samples were evaluated with the flow cytometry for lymphocyte subsets and Tr cells. The results showed that the proportion of CD3(+) and CD4(+) T cells, and the ratio of CD4/CD8 in patients with B-NHL group was significantly less than those in the healthy controls, i.e. (68.33 +/- 15.27)% versus (72.06 +/- 9.26)%; (34.47 +/- 12.84)% versus (42.45 +/- 9.2)%; 1.36 +/- 0.26 versus 1.92 +/- 0.20, but the prevalence of the CD4(+) CD25(high) Tr cells was significantly higher than those in the healthy group [(4.10 +/- 1.21)% versus (2.04 +/- 1.03)%, P < 0.001]. The ratio of CD4/CD8 in chemotherapy group was lower than that in control, but the proportion of CD4(+) CD25(high) Treg cells in chemotherapy group was higher than those before chemo-/radio-therapy and the control. It is concluded that the relative increase of CD4(+) CD25(high) Tr cells in peripheral blood of B-NHL patients may be related to immunosuppression and tumor progression.

[Effects of D-limonene on leukemia cells HL-60 and K562 in vitro].

Abstract: To investigate the effects of D-limonene (D-L) on the cell growth and apoptosis in HL-60, K562 cells and to elucidate its mechanism, the influence of D-L on proliferation of HL-60 and K562 cells was determined by propidium iodide assay, the expression levels of mutant p53, bcl-2, bax gene were detected by cell morphological analysis, flow cytometry and immunohistochemistry staining, the D-L-inducing HL-60 and K562 cell apoptosis in vitro was observed systematically. The results showed that D-L inhibited HL-60 and K562 cell growth in a dose- and time-dependent manner with the IC50 of 0.75 mmol/L similarly, D-L induced apoptosis of HL-60 and K562 cells, and expression of bcl-2 gene was down regulated by D-L in a concentration-dependent manner in HL-60 cells. The bcl-2, mutant type of p53 genes were down regulated while bax gene was up regulated by D-L in a concentration-dependent manner in K562 cells. It is concluded that D-L can inhibit proliferation and induce apoptosis of HL-60 and K562 cells. The bcl-2, mutant type of p53 and bax may be involved in the gene regulation of D-L-induced apoptosis.

[Mechanism of G2/M blockage triggered by activated-Chk1 in regulation of drug-resistance in K562/A02 cell line].

Abstract: The study was purposed to investigate the effect of phosphorylated-chk1 on cell cycle and apoptosis of human erythroleukemic cell line K562 and K562/A02, and to explore the mechanism of chk1 in regulation of drug-resistance of leukemia cells After treatment with adrimycin for six hours, the cell cycle distribution was detected by flow cytometry; the Chk1mRNA expression was detected by RT-PCR and the Chk1 phosphorylation level was detected by Western blot Under the condition of down-regulation of Chk1mRNA expression in cells transfected with Chk1 short hairpin RNA, the cell apoptosis rates were detected by flow-cytometry following adrimycin The results indicated that the proportion of K562/A02 cell line in G2/M phase was (5412 +/- 057)% at 6 hours after drug treatment, significantly higher than that of K562 cell line (3699 +/- 128)% No evident difference of the Chk1mRNA expression was observed between K562 and K562/A02 cell lines, while elevated Chk1 phosphorylation following DNA damage induced by adriamycin was observed in the K562/A02 cell line (079 +/- 056), significantly higher than that in K562 cell line (027 +/- 147) The cell apoptosis rate of the Chk1 shRNA group in K562/A02 cell line was 384-fold of blank vector group, but that in K562 cell line was 130-fold of blank vector group It is concluded that the increased chk1 activity that delay the progress of cell cycle are associated with cellular resistance to adrimycin in the K562/A02 cell line

[Indoleamine 2, 3-dioxygenase activity in acute myeloid leukemia cells contributing to tumor immune escape].

Abstract: This study was aimed to investigate the mechanism of indoleamine 2, 3-dioxygenase (IDO) activity in acute myeloid leukemia cells contributing to tumor immune escape Myeloid leukemia cells were isolated from bone marrow of 23 patients with acute myeloid leukemia (AML) and IDO expression was detected by immunochemistry and RT-PCR methods Then mixed lymphocyte reaction (MLR) of one way was carried out, leukemia cells were used as stimulating cells and T-lymphocytes were used as reactive cells in culture with or without 1-MT T-lymphocyte proliferation rate was determined by MTT assay and IDO activity in supernatant of MLR was detected by high-performance liquid chromatography (HPLC) The results showed that IDO expression was found in 17 out of 23 cases of acute myeloid leukemia cells; IDO enzyme activity in leukemia cells inhibited T-lymphocyte proliferation in MLR cultures It is concluded that IDO activity expressing in leukemia cells can suppress T-lymphocyte proliferation responses, which may be contributing to tumor immune escape

[Expression of SDF-1alpha and its receptor CXCR4 in acute leukemias and their relationship with extramedullary infiltration].

Abstract: The study was aimed to explore the expression of stromal cell derived factor-1alpha (SDF-1alpha) and its receptor CXCR4, and their relationship with the extramedullary infiltration in acute lymphoblastic, grannulocytic and monocytic leukemia. 66 cases of acute leukemia included 31 cases of acute lymphoblatic leukemia (ALL), 20 cases of acute grannulocytic leukemia (M(2)) and 15 cases of acute monocytic leukemia (M(4)+M(5)). There were 41 cases with extramedullary infiltration and 25 cases without-extramedullary infiltration. Enzyme-linked immunoabsorbent assay (ELISA) and flow cytometry were used to determine expression of SDF-1alpha and CXCR4 respectively on leukemia cells in peripheral blood and bone marrow of different groups. The results showed that average plasma level of SDF-1alpha in the ALL, M(4)+M(5), M(2) patients and the normal control were 1317.87 +/- 220.76, 1339.79 +/- 187.06, 1063.70 +/- 190.74, 1908.34 +/- 135.55 (pg/ml) respectively. The average levels in the ALL, M(4)+M(5) and M(2) patients groups were lower than those in normal control group. Both levels in ALL and M(4)+M(5) patient groups were higher than that in M(2) patient group. The average levels of SDF-1alpha in patient group with extramedullary infiltration and patient groups without-extramedullary infiltration were 1252.49 +/- 263.12, 1234.91 +/- 185.50 (pg/ml) respectively. The former seemed as if higher than the latter, but without statistical significance. The MFI of CXCR4 expression in ALL, M(4)+M(5), M(2) patient group were 78.47 +/- 33.96, 67.21 +/- 24.29, 41.66 +/- 17.18, respectively. CXCR4 expression in ALL and M(4)+M(5) patient groups were higher than that in M(2) patient group (P > 0.05). There was no significant difference between the ALL and M(4)+M(5) patient group (P > 0.05). The MFI of CXCR4 expression in patients with extramedullary infiltration and patients without extramedullary infiltration were 81.72 +/- 27.63, 36.94 +/- 11.86 respectively. The former was higher than the latter (P < 0.05). It is concluded that the higher expression of CXCR4 on acute lymphoblatic and monocytic leukemia cells may be one of the molecular mechanisms of extramedullary infiltration in both kinds of leukemia. The average plasma levels of SDF-1alpha decreased in leukemia patients and this decrease not related to the extramedullar infiltration, which may be due to the SDF-1alpha local expression in the organ infiltrated.

Application of CD123 in detection of minimal residual disease in acute promyelocytic leukemia

Abstract: The study was aimed to investigate the role and significance of CD123 with other immunological markers in detecting minimal residual disease (MRD) of APL patients. The immunophenotypes of 186 newly diagnosed APL patients and the percentages of cells identical with APL cell immunophenotypes in 20 normal bone marrow samples were analyzed using four-color flow cytometry. MRD in 172 specimens were monitored by mainly using CD34/CD117/CD123/HLA-DR four-color antibody panels, meanwhile 18 specimens were analyzed with the second antibody combination: CD9/CD117/CD34/CD33, simultaneously and the results were compared with real-time PCR. One hundred and sixteen of 172 bone marrow (BM) or peripheral blood (PB) specimens were from follow-up 19 newly diagnosed APL patients and the rest 56 samples were from 47 patients treated 3 to 24 months later. Among them, 117 samples and 55 samples were collected after achieving morphologic complete remission (mCR) and before achieving mCR respectively. The results of immunophenotyping demonstrated that except CD9, CD33 and CD117 were high-expressed and CD34 and HLA-DR were rarely expressed, the CD123 was expressed in 30/30 (100%) APL patients. The percentages of CD117(+)CD34(-)CD123(+)HLA-DR(-) and CD117(+)CD34(-)CD9(+)CD33(+) cells in nucleated cells were 0.066% +/- 0.012% and 0.089% +/- 0.066% in 20 normal bone marrow samples. The median time of achieving morphology complete remission in 19 APL patients was 4 weeks (3 - 6 weeks). The median time of FCM and PCR results turned to be negative in 13 APL patients was 7.5 weeks (5 - 11) and the median time of PCR results turned to be negative in 11 APL patients was 8 weeks (5 - 12). 41/117 (35.04%) samples were MRD positive by FCM after achieving mCR. The ratio of CD117(+)CD34(-)CD123(+)HLA-DR(-) cells was 5% in another 8 specimens, their median percentages of CD117(+)CD34(-)CD123(+)HLA-DR(-) cells were 0.48% (range 0.02% - 4.70%) and 9.02% (range 5.26% - 18.14%) respectively. The median relative percentages of CD123(+)HLA-DR(-) cells in CD117(+)CD34(-) population were 63.59% (range 15.11% - 98.36%) and 86.77% (range 63.29% - 92.62%) respectively. In FCM MRD positive samples, 95.9% (93/97) were PCR positive, the false positive rate of FCM and the false negative rate of PCR were 4.1% (4/97) and 8.75% (7/93) respectively. In FCM negative samples, 92% (69/75) were PCR negative and 8% (6/75) were PCR positive. The percentages of CD117(+)CD34(-)CD123(+)HLA-DR(-) cells in 116 consecutive specimens and 117 specimens of mCR were related to PML/RARalpha quantified by real-time PCR (r = 0.824, P < 0.001 and r = 0.754, P < 0.001 respectively). It is concluded that the detection of APL patients by means of two sets of antibody panels is simple and suitable, which is complementary to PCR in monitoring MRD of APL patients.

Expression and Significance of X-linked Inhibitor of Apoptosis Protein and Its Antagonized Proteins in Acute Leukemia

Abstract: To investigate the expression and significance of X-linked inhibitor of apoptosis protein (XIAP) and XIAP-associated factor 1 (XAF1) in acute leukemia, the expression of XIAP, XAF1, Smac, and HtrA2 mRNA in the bone marrow aspirates from 87 newly diagnosed AL patients, 23 patients in remission, 6 patients in relapse, and 17 normal controls were detected by means of reverse transcriptase polymerase chain reaction (RT-PCR), and their relationship with clinical therapeutic efficiency was analyzed. The results showed that the expression level of XIAP mRNA in newly diagnosed AL patients (0.990 +/- 0.337) was significantly higher than that in normal controls (0.395 +/- 0.148) (P < 0.01); the positive rate and expression level of XAF1 mRNA in newly diagnosed AL patients (56.32%, 0.246 +/- 0.267) were significantly lower than that in normal controls (100%, 0.964 +/- 0.387) (P < 0.01). In 69 out of 87 newly diagnosed AL patients, efficacy remained evaluable. AL patients with high level of XIAP achieved a lower complete remission (CR) rate than patients with low level of XIAP (54.55% and 86.11%, respectively) (P < 0.01). XAF1 positive patients achieved a higher CR rate than XAF1 negative patients (86.84% and 51.61%, respectively) (P < 0.01). It is concluded that the overexpression of XIAP and negativity of XAF1 may be two adverse prognostic factors in AL patients.

Human fetal heart-derived adherent cells with characteristics similar to mesenchymal progenitor cells.

Abstract: This study was aimed to investigate if human heart harbored a population of primitive undifferentiated cells with the characteristics of MPC. Cells were isolated from human fetal heart and were cultured under conditions appropriate for bone marrow-derived MPCs. Their morphology, phenotypes and functions were tested by methods developed for MPC from other sources. The results showed that morphologically, cells were spindle shaped and resembled fibroblasts. In their undifferentiated state, cells were CD73, CD105, CD29, CD44, HLA-ABC, CD166 positive and CD45, CD34, CD86, HLA-DR negative. When cultured in adipogenic, osteogenic or chondrogenic media, cells differentiated into adipocytes, osteocytes and chondrocytes respectively. They could be extensively expanded in vitro and exhibited very low immunogenicity as evaluated by T cell proliferation assays. It is concluded that cells isolated from fetal heart possess similarity to their adult and fetal bone marrow counterparts in morphologic, immunophenotypic, and functional characteristics.

[Overexpression of hypoxia inducible factor-1alpha (HIF-1alpha) promotes the differentiation of endothelial progenitor cell ex vivo].

Abstract: To investigate the influence of HIF-1alpha overexpression on the differentiation of endothelial progenitor cells (EPCs) ex vivo, EPCs were isolated from human peripheral blood by density gradient centrifugation, overexpressed HIF-1alpha was transfected to EPCs by electroporation; HIF1alpha, HIF1beta, vascular endothelial growth factor (VEGF) mRNA level were measured with RT-PCR; HIF-1alpha protein was detected with immunohistochemistry in a time course CD31(+) cells were measured with flow cytometry Cell morphology was observed after transfection The results showed that the transfection efficiency of HIF-1alpha to EPCs was about 20% HIF-1alpha and its controlled target gene VEGF were markedly induced by HIF-1alpha vector (P 005) HIF-1alpha protein was induced by HIF-1alpha transfection after 12 hours but was undetectable at 24 hours After 7 - 14 days cultured in 21% oxygen pressure, fluorescence-trace experiments revealed that CD31 + EPCs/EC could be generated more efficiently from overexpressed HIF-1alpha than that from pEGFP transfected group (P > 005) EPC morphology was observed by light microscopy HIF-1alpha-transfected cells under normoxia sprouted more rapidly from the EPC colonies than the untransfected cells or cells transfected with an GFP vector, which essentially maintained the original colony formation HIF-1alpha transfected cells took on an array-like arrangement rather than random dispersal, suggesting that they were in an advanced state of differentiation It is concluded that the utility of overexpression of HIF-1alpha can induce target genes which have influence on cell differentiation HIF-1alpha transfection was found to give a prospected way to do the insight research on ischemic treatment in vivo

[Expression of apoptosis-related proteins in the human bone marrow hematopoietic cells treated by Panax Notoginosides].

Abstract: The study was aimed to investigate the action of Panax Notoginosides (PNS, extracted from notoginseng herb) on the expression of the apoptosis-related proteins (Daxx, Fas) and transcription factors (NFkB, c-Rel) in the hematopoietic cells and to explore the mechanisms of supporting cells to survive. The colony formation of CFU-GM and CFU-E in human bone marrow was assayed in the presence of various concentrations of PNS. The viability of cells was assayed by trypan blue and the changes of cell morphology were observed with microscope. The Annexin-V positive cells were detected by FCM. Three lineages of human myeloid HL-60, erythroid K562, megakaryoid CHRF-288 and Meg-01 cells were incubated in addition of PNS (10 mg/L) for 14 days. The nuclear or cytoplasm protein of cells was extracted and analyzed by Western blot with monoclonal antibodies against Daxx, Fas or NFkB, c-Rel. The results showed: (1) the proliferation on hematopoietic progenitor cells (CFU-GM and CFU-E) and four cell lines was promoted by PNS; (2) after the four cell lines were promoted by PNS and hungered through wiping off the sera, the viability of the four cell lines was high without significant morphological change and neither the detection of Annexin-V positive cells; (3) the expression of Daxx and Fas protein could be inhibited by PNS. Western Blot showed that Daxx in four cell lines treated by PNS were 33.3-61.5% lower than that in untreated controls. The Fas protein was also descended in three cell lines of K562, CHRF-288 and Meg-01 by 33.3-71.4% respectively, while Fas protein in HL-60 cells was no detectable difference after PNS treatment. (4) The transcription factors NFkB and c-Rel protein could be increased by PNS. The NFkB, c-Rel protein were also enhanced in three cell lines of K562, CHRF-288 and Meg-01 by (2.0-2.7) and (1.5-2.3)-fold respectively, while there were also no detectable difference in HL-60 cells after PNS treatment. It is concluded that PNS inhibites the expression of Daxx and Fas proteins, may decrease the apoptosis of the hematopoietic cells. The level of NFkB and c-Rel proteins can be enhanced by PNS, which not only stimulates the proliferation of cells, but also inhibits the activity of the waterfall of caspase and apoptosis of the hematopoietic cells. PNS may treat the disease with over-apoptosis of hematopoietic cells, as aplastic anemia.

[Studies on ex vivo expansion of megakaryocytic progenitor and its application--review].

Abstract: Application of ex vivo expanded megakaryocytic progenitor cells (MKPC) is a strategy for the treatment of thrombocytopenia after hematopoietic stem cell transplantation Some growth factors including thrombopoietin (TPO), megakaryocyte growth and development factor (MGDF), interleukin (IL)-1, IL-3, IL-6, IL-11, platelet-derived growth factor (PDGF), and serotonin (5-HT) have been demonstrated to play an important role on the regulation of megakaryocyte/platelet development, the efficient conditions for the expansion of the megakaryocyte (MK) progenitors from hematopoietic stem/progenitor cells were discussed in this review article TPO alone produced a high proportion of MK progenitors but a low total cell count The addition of IL-1 beta, IL-3, IL-6 and Flt-3L improved the expansion outcome The combination of three to five cytokines produced more efficient expansions of hematopoietic stem and MK progenitors PDGF also enhanced the ex vivo expansion of CD61+ CD41+ cells and CD34+ cells in combination with TPO, IL-1 beta, IL-3, IL-6 and Flt-3L PDGF is a suitable growth factor to improve the ex vivo expansion of MKPC without promoting their in vitro maturation More importantly, PDGF also enhanced the engraftment of human stem and progenitor cells in NOD/SCID mice It has been reported that MKPC can be safely administered to autologous peripheral blood progenitor cell transplant recipients In short, MKPC can be expanded ex vivo and safely applied to autologous transplant

Combined effect of recombinant mutant human TRAIL and daunorubicin in inducing apoptosis of leukemia cell and its mechanism

Abstract: The aim of study was to investigate the combined effect of recombinant mutant human TRAIL (rmhTRAIL) with daunorubicin (DNR) or alone on K562 and U937 leukemia cell lines and its mechanism. The fibroblasts (MRC-5) of normal-human embryonic lung were used as control cells. After being treated with rmhTRAIL and DNR or only with rmTRAIL, the cytotoxic effect and the apoptosis rate in K562, U937 cells were measured by MTT assay. The expression levels of TRAIL death receptor and TRAIL decoy receptor mRNA in these three cell lines were assayed by semiquantitive RT-PCR before and after treatment with DNR. The results indicated that K562 and U937 were sensitive to rmhTRIAL. DNR had synergistic inhibitory effect with rmhTRAIL on the growth of K562 and U937 cell lines (P < 0.05). The expression level of DR4 and DR5 mRNA was significantly higher in K562 and U937 with combined treatment of rmhTRAIL and DNR than that in those alone, while the expressions of DcR1 and DcR2 mRNA were not influenced. It is concluded that in vitro, rmhTRAIL alone or in combination with DNR can obviously inhibit the growth of leukemia cell lines and induce cell apoptosis, DNR and rmhTRAIL have a synergistic inhibitory effect on growth of K562 and U937. The mechanism may correlate with the up-regulation of DR4 and DR5 of K562 and U937.

[Deferoxamine induces apoptosis of HL-60 cells by activating caspase-3].

Abstract: This study was purposed to observe the changes of caspase-3 activity during apoptosis of HL-60 cells induced by an iron chelator, DFO (deferoxamine), and to explore the mechanism underlying apoptosis in HL-60 cells. The HL-60 cells treated with DFO were examined by light microscopy, flow cytometry (FCM) and DNA agarose gel electrophoresis; the activity of caspase-3 was determined by cellular immunohistochemistry; the transcription of the apoptotic gene of bax was detected by hybridization in situ. The results showed that the typical morphological character of apoptosis cells, DNA ladder and FCM assay confirmed that DFO could induce the apoptosis in HL-60 cells. The apoptotic rate increased in dose-and time-dependent manner. When cells had been cultivated with 100 micromol DFO for 12 hours, a few caspase-3 positive cells were found. In the process of time, the rate of caspase-3 positive cells was progressively higher than that in control (P < 0.05), while the level of bax transcription was also higher than that in the control. It is concluded the activation of caspase-3 and gene bax may be involved in the apoptosis of HL-60 cells induced by DFO.

Inhibitory effect of curcumin on angiogenesis induced by brain derived neurotrophic factor from multiple myeloma cells

Abstract: In order to explore the probability of curcumin treating multiple myeloma (MM) via the inhibition of angiogenesis, the expressions of brain derived neurotrophic factor (BDNF) and its specific receptor in human MM cells and endothelial cells were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The angiogenic activity was evaluated by endothelial cell migration assay and tubule formation assay in vitro. The results showed that exogenous BDNF significantly induced endothelial cell tubule formation and endothelial cell migration, these two effects were inhibited by curcumin. Furthermore, BDNF was detected in the MM cell and TrkB was detected in the endothelial cell and curcumin depressed the mRNA expression of BDNF and TrkB in the dose- and time-dependent manners. It is concluded that BDNF is a novel angiogenesis protein. Curcumin interrupts the interaction between multiple myeloma cells and endothelial cells by reducing TrkB expression in endothelial cells and inhibiting BDNF production in multiple myeloma cells, eventually, resulting in inhibition of angiogenesis. This is probably one part of the mechanism of the curcumin treating MM via the inhibition of angiogenesis.

Leukemia-Associated Immunophenotypes in 415 Childhood and Adult Patients with B Lineage Acute Lymphoblastic Leukemia by Multiparametric Flow Cytometry Analysis

Abstract: To evaluate the significance of FCM in minimal residual disease (MRD) detection, the immunophenotyping and leukemia-associated immunophenotypes (LAIP) of leukemia cells from 273 adult and 142 childhood patients with B lineage acute lymphoblastic leukemia (B-ALL) were detected by four to six antibody combinations of 4-color CD45/SSC gating multiparametric flow cytometry (FCM). The results showed that the B-ALL patients could be classified into 4 subtypes based on different expression CD34 and CD10: subtype I (CD34(+)/CD10(-)), subtype II (CD34(+)/CD10(+)), subtype III (CD34(-)/CD10(+)), subtype IV (CD34(-)/CD10(-)). The LAIP was observed in 100% and 92% patients of subtype I and subtype II, respectively, whereas only 79.2% in subtype III. The incidence of LAIP in total B-ALL cases was 90% by using the antibodies detected in this investigation. There was no significantce different for incidence of LAIP between adult and pediatric patients. LAIP was observed in 77.6% of patients by labeling only CD34/CD10/CD19/CD45 4-color antibody combination. It is concluded that in 90% of childhood and adult B-ALL patients LAIP can be found, which suits MRD detection by multiparameter flow cytometry.

Genetically modified myeloma cell vaccine inducing antitumor immune response in vivo.

Abstract: This study was aimed to evaluate the in vivo antitumor effect of genetically modified myeloma cell vaccine on human myeloma xenografts implanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Human immune system was established in NOD/SCID mice by intraperitoneal injection of human peripheral blood lymphocytes (PBLs). After being inoculated subcutaneously with irradiated myeloma cell line sko-007, adenovirally transferred with GFP or p53, granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7-1 genes, huPBL-NOD/SCID mice were challenged by subcutaneous injection of non-transferred sko-007 cells. The results indicated that Ad-p53/GM-CSF/B7-1-infected sko-007 cell vaccination significantly reduced local tumor growth compared with controls. Histopathological and immunohistochemical analysis showed that tumor tissues increasingly displayed diffuse necrosis, mainly caused by apoptosis, accompanied with significant fibroplasias and blood vessel hyperplasia, and human T cells infiltrated into the tumor tissues. It is concluded that transgenic p53, GM-CSF and B7-1 expression produces an immune response against myeloma cells and may be of therapeutic value for multiple myeloma in human being.

Molecular basis of partial D phenotypes in Chinese

Abstract: To investigate the molecular basis of partial D phenotypes in Chinese, D variants with weak D expression was screened by using indirect anti-human globulin test (IAT) method, the polymerase chain reaction-sequence specific primer (PCR-SSP) method was employed to amplify RHD specific exons and their flanking regions. The amplification products were sequenced directly to determine the molecular basis of D variants. The results showed that ten cases of partial D phenotypes, including one case of D Va (Kou.), one case of D Va (Hus.), one case of D Va-like (YH.), and seven cases of D VI type III, were detected from 22 cases of weak D phenotype respectively. All ten cases of partial D phenotypes had one RHD allele deleted. In conclusion, the molecular basis of ten cases of partial D phenotype was confirmed, including D Va (Kou.) and D Va-like (YH.) phenotypes reported firstly in Chinese population.

Immunophenotypic features in leukemia of NK cell series

Abstract: The aim was to investigate the immunophenotypes of NK series leukemia. Immunophenotypes of 297 cases of acute leukemia (AL) were measured by flow cytometry, and these immucopenotypic features were analyzed. The results showed that 43 out of 297 cases of AL (14.5%) were CD56 positive. 6 cases were NK series leukemia and 37 cases were acute myelogenous leukemia with CD56 expressed. One patient has been diagnosed as myeloid/NK cell precursor acute leukemia, two patients were blastic NK cell leukemia, one was supposed to be NK-like T-cell lymphoma/leukemia, while another one was large granular lymphocyte leukemia (LGLL). It is concluded that almost all of CD56 positive leukemia were acute myelogenous leukemia with CD56 expressed. The immunophenotypes of NK series leukemia were antigens from hematopoietic stem cells to T/NK progenitor cells with meyloid antigen positive, and through NK progenitors to mature NK cells. The immunophenotypes of heterogeneous NK leukemia cells are different, that should be carefully distinguished.

Biological Properties of Mesenchymal Stem Cells Derived from Bone Marrow of Patients with Multiple Myeloma

Abstract: The study was purposed to explore the role of mesenchymal stem cells (MSCs) in the pathogenesis of bone disease particularly observed in multiple myeloma (MM), the biological features of marrow derived MSCs from patients with MM have been investigated. Marrow aspirates were harvested from 11 newly diagnosed patients with MM and 5 normal adults and MSCs were isolated and culture-expanded by the cell properties of adherence to plastic flasks, The phenotype was analyzed by flow cytometric technique. The proliferation of MSCs was observed by MTT assay and their differentiation capacities into osteoblasts and adipoblasts were assessed with lineage-specific histochemical staining. The concentrations of IL-6 and SCF in the culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). MSC culture supernatants were collected and MTT assay was performed to evaluate their support on the proliferation of an MM cell line SKO007 cells. The results showed that bone marrow-derived MSCs from MM patients were homogeneously positive for CD29, CD73, CD166 and HLA-ABC and negative for hematopoietic cell marker CD45 and endothelial cell marker CD31, the phenotype of which was similar to that of marrow counterparts from normal adults. MTT assay indicated that MSCs from MM patients or normal adults proliferated at similar rates. MSCs from MM patients occupied in vitro osteogenic and adipogenic capacity as those from normal adults. The levels of IL-6 and SCF in culture supernatant were greatly up-regulated in MM patients by ELISA assay. Furthermore, MSC culture supernatants from MM bone marrow displayed enhanced activity to promote the proliferation of SKO007 cells. It is concluded that marrow-derived MSCs from bone marrow of MM patients are normal in their proliferation and differentiation capacities, and myeloma bone disease may not be ascribed to the differentiation of MSCs while the elevated secretion of IL-6 and SCF may provide necessary cues for the survival of malignant myeloma cells.

[Effects of rhG-CSF mobilization on immunological properties of grafts from peripheral blood and bone marrow].

Abstract: This study was aimed to investigate the difference of immunological properties between recombination human granulocyte colony-stimulating factor (rhG-CSF) mobilized peripheral blood grafts (G-PB) and rhG-CSF primed bone marrow grafts (G-BM). The lymphocyte proliferation ability and the quantities of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) secreted by T cells were determined by using MTT assays and sandwich ELISA; T cell subgroups, dendritic cells (DC), monocytes and the expression of CD28 costimulatory molecules on T cells were determined by multicolor flow cytometry. The results showed that the absolute numbers of lymphocytes, monocytes, CD3+, CD4+ and CD8+ T cells as well as DC1 and DC2, the ratios of CD4/CD8 in G-PB were significantly higher than those in G-BM, respectively (P < 0.001). T cell proliferation ability was significantly higher in G-PB than that in G-BM (P < 0.05). The quantities of IFN-gamma and IL-4 secreted by T cells per microliter of G-PB was significantly higher than those of G-BM, the ratios of IL-4/IFN-gamma were significantly lower in G-PB than that in G-BM (P < 0.001). As compared with G-BM, the ratio between DC2 and T-lymphocyte was significantly low in G-PB (P < 0.01), whereas the percentage and overall expression of CD28 on CD4+ and CD8+ cells were significantly high in G-PB (P < 0.05). It is concluded that T cell hyporesponsiveness of G-PB and G-BM induced by rhG-CSF in vivo were confirmed to be different, and the difference of immunological properties between G-PB and G-BM may explain the lower incidence of GVHD and lower relapse after G-BM and G-PB transplantation respectively.

[Difference between FOXP3 gene expressions in donor grafts with or without acute graft-versus-host disease].

Abstract: The study was aimed to investigate the association of FOXP3 gene expression in donor grafts with acute graft-versus-host disease after HLA-identical sibling allogeneic hematopoietic stem cell transplantation. Twenty-six donor grafts (peripheral blood or bone marrow) and their respective clinical characteristics were evaluated. Flow cytometry analysis was performed to assess the percentage of CD4+CD25+ and CD4+CD25(high) T cells in cord blood, healthy controls' peripheral blood and donor grafts. Relative transcripts of FOXP3 mRNA were determined by real-time quantitative reverse transcription -polymerase chain reaction with beta2-MG as the internal control gene. The specificity of FOXP3 and beta2-MG amplifications was confirmed by analyzing the dissociation curves and electrophoresis of the target amplicon. The results showed that the CD4+CD25+ T cells in peripheral blood, peripheral blood stem cell (PBSC) or BM grafts exhibited a continuous and primarily low expression of CD25 and the frequencies of CD4+CD25+ T and CD4+CD25(high) T in CD4+ T cells were (48.5 +/- 16.3)% and (9.6 +/- 2.5)%, (42.1 +/- 14.7)% and (13.1 +/- 4.2)%, (43.4 +/- 9.6)% and (14.6 +/- 4.5)%, respectively. There was no significant difference in the frequencies and absolute numbers of CD4+CD25(high) T cells between patients with aGVHD and patients without aGVHD (P > 0.05). The plot of log transfused cDNA amount versus DeltaCt had a slope of 0.0826 which indicated approximately equal efficiency of FOXP3 and beta2-MG amplifications in real-time PCR. The specificities of amplification were confirmed by analyzing the dissociation curves and electrophoresis of PCR products with the values of Tm 86.5 degrees C and 82.3 degrees C, respectively. The relative transcripts of FOXP3 in PBSC grafts of recipients without aGVHD were 318%high as those with aGVHD (median of 41.0 x 10(-5) and 12.9 x 10(-5), respectively) (P = 0.03). No significant difference was found in other related variables for GVHD. It is concluded that coexpression of CD4 and CD25 may be insufficient to identify regulatory T cells; FOXP3 mRNA expression may be specifically quantified with real-time quantitative RT-PCR using SYBR Green I chemistry. FOXP3 mRNA expression in donor grafts is significantly low in patients with aGVHD compared with patients without aGVHD. It indicated that the expression level of FOXP3 mRNA may be one of the useful indicators for in predicting aGVHD.

Modified guanidine hydrochloride method for DNA extraction from cord blood used in HLA genotyping

Abstract: To compare two different methods for extracting genomic DNA from cord blood and to evaluate their applications for HLA genotyping, the genomic DNA from 72 samples was extracted by guanidine hydrochloride (Gu * HCl) and modified guanidine hydrochloride, the DNA yield and purity were evaluated by spectrophotometry and detected by PCR with sequence-specific primers The result showed that the genomic DNA was successfully isolated from whole blood by both methods The modified Gu * HCl method used was better than Gu * HCl method as the modified method produces better quality of DNA and less ambiguous bands in PCR It is concluded that modified Gu * HCl method has the advantages of low-cost, simple operation, high quality output and clear positive bands in HLA-genotyping, the modified method is optimal for extracting DNA from multiple samples of cord blood bank

[HSC transplantation-associated intestinal thrombotic microangiopathy: clinical pathological features, diagnosis criteria and treatment].

Abstract: Thrombotic microangiopathy (TMA) is a lethal transplantation-associated complication which exactly likes acute intestinal graft-versus-host disease (GVHD) in the clinical manifestation. 373 consecutive patients with hematological diseases received family HLA matched or mismatched HCT from May, 2002 to July, 2004. To analyse the clinical and pathological characteristics of TMA, 30 patients who suffered from severe diarrhea and received colonoscopic examination and gut biopsy were retrospectively analyzed. The results indicated that 7 patients originally diagnosed as gut GVHD showed the pathological evidence of enteric TMA. The incidence of TMA was 7 out of 30 specimen (23.3%). Pathological evidence of enteric TMA shown microvascular disorder characterized by thrombus in the capillary without infiltration of lymphocytes and perivascular hemorrhages in the mucosa, swelling and focal denudation of epithelial cells. All patients with TMA were associated with cytomegalovirus (CMV) antigenemia/disease. Among these patients, 4 cases, who only showed TMA without the evidence of gut GVHD pathologically, displayed treatment-resistant bloody diarrhea, renal failure, veno-occlusive disease, hemorrhagic cystitis, hemolytic anemia as well as thrombocytopenia. But the other 3 cases, with co-existence of both TMA and GVHD pathological characteristics had better treatment response. Survival analysis indicated that 3 patients with TMA-GVHD survived for 461 to 536 days but three out of four TMA patients died from VOD with liver failure as well as multiple organ failure during 101 to 254 days after HCT. In conclusion, to better diagnose those patients with severe and refractory diarrhea following HCT, pathological examination may indicate crux evidence to identify intestinal TMA from gut GVHD. Furthermore, this primary report has first evidenced that TMA and TMA-GVHD are two pathologically well-recognized subtypes with the difference between the pathological characteristics, treatment response and clinical outcomes.