To explore the regulatory effects of Egr-1 promoter sequences in transcriptional targeting by 5-fluorouracil(5-Fu) on the expression of hematopoietic growth factor genes.

To investigate the effects of enamel matrix proteins (EMPs ) on proliferation, alkaline phosphate (ALP) activity of bone marrow stromal cells (BMSCs) seeded on the scaffold of chitosan thermosensitive hydrogel.

To establish an in vitro long-term culture system of mouse Spermatogonial stem cells (SSCs).

To investigate the morphological and quantitative alterations of peripheral blood endothelial progenitor cells (EPCs) in patients with ST elevation myocardial infarction (STEMI) after snake venom fibrinolytic enzyme therapy.

To investigate the changes in peripheral blood bone marrow stem cells and tumor necrosis factor-alpha gene expression in the ischemic myocardium in rabbit models of hibernating myocardium.

To assess the differentiation potential of rat adipose-derived stem cells (ADSCs) into Schwann-like cells in vitro.

To study the compatibility between human adipose-derived mesenchymal stem cells and porcine bone scaffolds.

To characterize the time course of spontaneous differentiation of in vitro cultured human embryonic stem cells (hESCs) into hematopoietic cells to provide experimental evidence for induction of hematopoietic commitment of hESCs.

ABCG2, a half-transporter concerning with the endo and exon-toxin-efflux, plays an important role in protecting the normal tissues from the toxin-hurt as well as mediating the multidrug resistance, because many of the chemotherapeutic drugs are the substrate of ABCG2. In this paper, the advance of research about this gene's single nucleotide polymorphisms (SNPs) was explained concisely. The relationship among ABCG2, the stem cells and the tyrosine kinase inhibitor (TKI) was reviewed. The research about drug resistance related-progress in hematologic malignancies was analyzed retrospectively and the present problems and the perspective in the future were discussed.

This study was purposed to explore the diagnostic role of flow cytometry in immunorelated pancytopenia (IRP). After 50 IRP patients were hospitalized, the concentration of serum ferritin, folic acid and vitamin B(12), immunologic test, platelet antibody, test of hepatitis A, B and C, haemolysis test and bone marrow smear examination were carried out, meanwhile the chromosome karyotype analysis and some routine examinations were performed. The 50 patients were divided into group A and group B. Group A consisted of 22 patients who were undefinedly diagnosed and intended to diagnosed as IRP, group B consisted of 28 definedly diagnosed patients with hematologic malignancies, including 7 cases of aplastic anemia, 2 of paroxysmal nocturnal hemoglobinuria, 10 of myelodysplastic syndrome, 9 of megaloblastic anemia. In addition, 30 normal people were used as normal control group (group C). For groups A and B, the binding autoantibodies of bone marrow stem/progenitor cells, erythroblasts and myelocytes were detected by flow cytometry, meantime the ratio of total B-(CD10(+)) and CD5(+) B-lymphocytes in peripheral blood was assayed. For control group, the ratios of CD19(+) and CD5(+) B lymphocytes in peripheral blood were determined alone. The results indicated that the detection of bone marrow autoantibodies in 20 patients of group A showed positive with 90.90%. The IgG type was found mostly in antibody binding types, next the IgM type, the IgA type was fewer. The detection of bone marrow autoantibodies of 2 patients in group B showed positive with 7.14%. The positive rate in group A was obviously higher than that in group B (p < 0.01). The ratios of CD19(+) and CD5(+) B lymphocyte in peripheral blood were significant higher in group A than that in group B and control group (p < 0.01), but there was no significant difference between groups B and control. It is concluded that the application of flow cytometry in detecting the autoantibodies of bone marrow cells and CD19(+) B-and CD5(+) B-lymphocyte in peripheral blood can provide reliable diagnostic evidence and detection measure for diagnosis and differential diagnosis of IRP, as well as may contribute to draw up more effective therapeutic strategy.

This study was aimed to investigate the specific anti-leukemia cell effect of cytotoxic T lymphocytes (CTLs) induced by HL-60 or K562 cell-sensitized dendritic cells (DCs) from umbilical cord blood. 12 units of human umbilical cord blood (UCB) were collected and the mononuclear cells (MNCs) were isolated from UCB, then cultured with granulocyte monocyte colony- stimulating factor (GM-CSF), interleukin 3 (IL-3), recombinant human stem cell factor (SCF) and EPO for 3 - 4 weeks. Flow cytometry was used to determine the number of DCs and cell surface antigens before and after culture with monoclonal antibodies including CD83, CD1a, CD11c and CDw123. HL-60 and K562 were frozen-thawed, and released their tumor antigen peptides (TAP). The CTLs were produced by sensitizing T lymphocytes with DC-loaded HL-60 and K562 cell antigens. The test of (3)H-TdR incorporation was used to detect the immunostimulation activity of DCs. MTT assay was applied to evaluate specific cytotoxicity of CTL on leukaemia cells. The results indicated that the MNCs of UCBs cultured with GM-CSF, IL-3, EPO and SCF were shown to differentiate into CD1a(+) CD11c(+) CD83(+) CDw123(+) DCs. Numbers of DCs from UCBs remarkably increased in 2 - 4 weeks and then decreased. After culture with cytokines DCs increased (10.6 - 28.2) x 10(5)/ml in actual numbers. The CTL induced by DC pulsed with HL-60, K562 frozen-thawed lysates were effective to kill HL-60 and K562. Cytotoxicity of CTL to HL60 and K562 were (42.04 +/- 8.46)% and (31.25 +/- 11.07)% respectively. It is concluded that the MNCs of UCBs cultured with cytokines of GM-CSF, SCF, EPO and IL-3 can differentiate into CD1a(+), CD83(+), CD11c(+) and CDw123(+) DCs. The CTL induced by DCs pulsed with HL-60, K562 frozen-thawed lysates can effectively kill HL-60 and K562. These DCs as antigen presenting cells play an important role in cancer immunotherapy.

This study was to investigate the reconstitution of NK cells and their receptors after unrelated cord blood stem cell transplantation (UCBT) and its clinical importance. 11 cases of acute leukemia underwent UCBT were enrolled in this study. The reconstitution of NK cells and their surface receptors as well as the the recovery of T and B cells within 90 days after clinical engraftment following UCBT were measured and analysed by flow cytometry. The results indicated that the recovery of NK cells appears to be relatively early. CD3(-)56(+) NK cell count was (35.12 +/- 18.66)% of peripheral blood (PB) lymphocytes on the day of clinical engraftment and higher than that in normal. The peak of the NK cells reached to (37.8 +/- 17.52)% of lymphocyte at 30 days after clinical engraftment. NK count was (30.4 +/- 19.14)% at 60 days after clinical engraftment when the absolute NK cell count reached to the peak (up to 544 cells/microl) in PB. The activated receptor NKG2D was reconstituted fast and high expressed [(79.58 +/- 8.71)%] at the time of clinical engraftment with a tendency of gradual elevation, which reached to peak value (82.55 +/- 9.10)% at day 60. Another activated receptor NKp46 also reconstituted fast, and maintained at a high level even at 90 days after clinical engraftment. The expression of NKG2A was lower than that of the activated receptor of NK cells, which tendency lasted for at least 90 days after clinical engraftment. The reconstitution of T cells in PB after UCBT was relatively slow with lower expression rate. It is concluded that the reconstitution of NK cells in patients with acute leukemia is earlier following UCBT. The earlier recovery of activated receptor of NK cells, especially NKG2D, suggests that the activation of NK cells may play a role in graft versus leukemia (GVL) effect in the early period after UCBT.

This study was purposed to investigate the effect of hematopoietic growth factor expression regulated by Egr-1 promoter on the recovery of hematopoietic function in bearing-melanoma mice after chemotherapy with doxorubicin (ADM). The human GM-CSF cDNA and enhanced green fluorescence protein (GFP) cDNA were linked together with internal ribozyme entry site (IRES) and then were inserted into the expression vector pCI-neo under control of the Egr-1 promoter (Egr-EG). This vector was transduced into human bone marrow stromal cell lines HFCL by lipofectamine and was transfused in severe combined immunodeficiency (SCID) mice. The experimental mice were randomly divided into 4 groups (6 mice in each group): (1) HFCL/EG + ADM group in which the HFCL/EG cells were transplanted intravenously in SCID mice bearing melanoma, ADM was given intraperitoneally after 3 days; (2) HFCL + ADM group in which the HFCL cells were transplanted intravenously, ADM was given intraperitoneally after 3 days; (3) HPCL/EG group in which HFCL/EG cells were transplanted alone; (4) HFCL group in which HFCL cells were transplanted alone. The dynamic change of peripheral blood picture was assayed by hemocytometer; the eGFP(+) human stromal cells were detected by flow cytometry; the expression of GM-CSF mRNA and protein were determined by RT-PCR and Western blot respectively. The results indicated that as compared with HFCL/EG and HFCL groups, the leukocyte count in HFCL/EG + ADM group decreased, but decrease level was weaker than that in HFCL + ADM group, meanwhile the recovery of leukocyte count was earlier than that in HFCL + ADM group. The CFU-GM amount between 4 groups showed no significant difference. The detection results showed that the inhibitory rate of tumor was related to chemotherapy, but not to expression of exogenous gene; the eGFP(+) stromal cells existed in bone marrow of mice treated with ADM. The RT-PCR and Western blot assays revealed enhancement of GM-CSF mRNA and protein. It is concluded that the ADM-inducible GM-CSF gene therapy regulated by Egr-1 promoter may promote the hematopoietic recovery after chemotherapy.

The aim of this study was to explore the difference of MSC migration mediated by SDF-1/CXCR4 axis through Boyden chamber in vitro migration assay. The SDF-1 density-dependence of MSC migration was observed. Subsequently, the effects of different blocking agents on hSDF-MSC migration were observed after MSC were treated with 50 nmol/L wortmannin, 10 micromol/L LY294002, 50 micromol/L PD98059, 10 micromol/L U73122, 126 micromol/L AMD3100 and 50 nmol/L verapamil respectively. The results showed the efficiency of MSC migration increased gradually with the increasing of hSDF-1 density. And after MSCs treatment with 50 nmol/L wortmannin, 10 micromol/L LY294002, 50 micromol/L PD98059, 10 micromol/L U73122 and 126 micromol/L AMD3100 respectively, the ability of MSC migration decreased. The ability of MSCs migration obviously decreased when MSCs were treated with U73122, AMD3100. It is concluded that the SDF-1/CXCR4-mediated MSC migration may be related to mitogen-activated protein kinase (MAPK), phosphatidylinositol phospholipase C (PI-PLC) and protein kinase (PKC) signal pathways.

This study was aimed to investigate the differentiation potential after EGFP gene-modified mesenchymal stem cells (MSCs) transfected by lentiviral vector (LV). After isolated, cultured and identified, MSCs were transfected with the lentiviral vector carrying EGFP gene in vitro. The transfection efficiency was measured by observing the expression of green fluorescence protein. At last, the transfected MSCs were induced into adipocytes in adipogenesis supplement medium, the induction level was detected by Sudan fat stain. The results indicated that after transfection for 72 hours, weak fluorescence in MSCs was observed under fluorescence microscope. After 21 days, many lipid droplets with high refractivity occurred in cytoplasm of transfected MSCs, and showed orange in Sudan black stain. There were no significantly differences between transfected and non-transfected cells (p > 0.05). It is concluded that MSCs were successfully transfected by LV carrying EGFP gene. The transfected MSCs maintain multiple differentiation and proliferation potential. In the adipogenesis supplement medium, transfected MSCs also can be induced to differentiate into adipocytes. MSCs can act as target cells for gene therapy.

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.

This study was purposed to investigate the effects of rat marrow mesenchymal stem cell (rMSC) transplantation on left ventricular (LV) function in a rat myocardial infarction model. Myocardial infarction was performed in male Lewis rats by ligating the proximal left coronary artery. Rats were randomly divided into 3 groups: sham operation group (only thoracotomy, n = 8), AMI group (DF12 injection, n = 10), rMSC group (Dil-Labeled rMSC transplantation). At 8 weeks later, the cardiac functions including left ventricular ejection fraction (LVEF), left ventricular end systolic pressure (LVESP), left ventricular end diastolic pressure (LVEDP), +dp/dtmax and -dp/dtmax were evaluated by echocardiography and cardiac catheterization. The presence and differentiation of engrafted cells were assessed. CD31 was detected by immunohistochemical staining to demonstrate neovascular formation. The results indicated that the cultured in vitro rMSC expressed CD90, CD44, CD105, CD54; did not express CD34, CD45, CD31, as compared with AMI group, rMSC group showed a significant increase of LVEF, LVESP, +dp/dtmax, -dp/dtmax and a significant decrease of LVEDP. Immunofluorescence demonstrated that some transplanted rMSCs were positive for myosin, suggesting that small number of transplanted rMSCs differentiated into cardiac-like cells. Immunostaining showed marked augmentation of capillary density in the rMSC group than that of AMI group. It is concluded that transplanted rMSCs can differentiate into cardiac-like cells and rMSC transplantation can improve LV function after myocardial infarction in rats.

This study was aimed to investigate abca5, mdr-1, kdr, dapk and irf-1 expressions in leukemia stem/progenitor cells (LSC) from CD7 positive acute myeloid leukemia, the expression of these 5 genes in mononuclear cells (MNC) from 15 normal bone marrow (NBM) and 16 AML patients bone marrow (AML BM) specimen were detected by real-time quantitative PCR (RQ-PCR). CD34(+)CD38(+) progenitor cells and CD34(+)CD38(-)Lin(-) stem cells were sorted by flow cytometry (FCM) from the MNCs of 10 NBM and 21 AML BM specimen. These 5 gene expressions in the sorted cells were detected by small amount cell RQ-PCR. The results showed that these 5 genes above mentioned all expressed in NBM-MNC, in which the expression levels of irf-1 and dapk were highest with the relative expression levels 4.08 and 3.86, the expression levels of abca 5 and mdr-1 were in the middle with the relative expression 0.49 and 0.84 respectively, the kdr expression was lowest with the relative expression level 0.02. In CD34(+)CD38(+) progenitor cells, the expression level of kdr increased dramatically (p < 0.05) while irf-1 and dapk dramatically decreased (p < 0.05). There was no obvious change of expression in the rest 2 genes. In CD34(+)CD38(-) stem cells the expression level of these 5 genes all increased nearly 2 times as much as that in CD34(+)CD38(+) progenitor cells, but kdr increased 3 times as much, and the increase of kdr and irf-1 expressions was of statistical significance (p < 0.05). Compared with the NBM, expression levels of 5 genes in AML-MNC decreased, and out of them abca 5, mdr-1, kdr and dapk were decreased most remarkably (p < 0.05). Comparison between AML CD34(+)CD38(+) cells and AML MNC showed that the expression level of irf-1 and dapk were decreased dramatically (p < 0.05) while the rest 3 genes increased their expression with statistical significance (p < 0.05). The expression levels of these 5 genes were higher in CD34(+)CD38(-) cells than those in CD34(+)CD38(+) stem cells, and the increase of kdr and irf-1 expressions showed statistical difference (p < 0.05). These 5 genes expression levels were all higher than those in CD34(+)CD38(+) cells whether in AML CD34(+)CD38(-)CD7(+) cells or CD34(+)CD38(-)CD7(-) cells. The increase of kdr expression in CD34(+)CD38(-)CD7(+) cells as well as kdr and irf-1 expressions in CD34(+)CD38(-)CD7(-) cells were all of statistical significance (p < 0.05). In conclusion the expression level of kdr in NBM was highest in stem cells while dapk and irf-1 were highest in differentiated cells. The expression levels of these 5 genes in CD34(+)CD38(-)Lin(-) stem cells were higher than those in CD34(+)CD38(+) progenitor cells. The gene expressions in AML CD34(+)CD38(-)CD7(+) cells and CD34(+)CD38(-)CD7(-) cells are in accordance with the characteristics of stem cells.

Bmi-1 is a transcriptional repressor, which belongs to the polycomb group family. It has been demon- started that over-expression of Bmi-1 occurs in a variety of cancers, including several types of leukemia. Bmi-1 gene plays a key role in regulation of self-renewal in normal and leukemic stem cells. Acute myeloid leukemic cells lacking Bmi-1 undergo proliferation arrest and show signs of differentiation and apoptosis, which leads to the proposal of Bmi-1 as a potential target for therapeutic intervention in leukemia. The purpose of this study was to investigate the effect of short hairpin RNA (shRNA) targeting Bmi-1 on functions of K562 cell line. The shRNA eukaryotic expression vector targeting Bmi-1 was constructed and transfected into K562 cells through lipofectamine 2000. The mRNA and protein levels of Bmi-1 were detected by PCR and Western blot respectively. The proliferation of K562 after Bmi-1 silencing was measured by using MTT assay and clone formation assay. The cell cycle was detected by flow cytometry. The results indicated that among the four shRNA designed, there was a shRNA which efficiently interfered with the expression of Bmi-1. The results of PCR and Western blot validated that the Bmi-1 gene of K562 cells transfected with such a Bmi-1 shRNA was suppressed successfully. Although levels of Bmi-1 mRNA and protein were significantly reduced, delivery of this siRNAs had no effect on cell viability or growth. Flow cytometry analysis suggested that Bmi-1 inhibition did not affect the cell cycle. It is concluded that the suppression of Bmi-1 expression is not able to reduce proliferation of K562 cells, suggesting existence of some other parallel signaling pathways, which are fundamental for leukemic transformation and are independent of Bmi-1 over-expression. Bmi-1 over-expression may play a secondary role in chronic myeloid leukemia transformation.