This study was aimed to investigate the effect of dexamethasone (Dex) on immunosuppressive ability of mesenchymal stem cells (MSC) during expansion and differentiation of MSC. MSC were cultured in 96-well flat-bottom plates. Proliferation assays were performed by using the BrdU colorimetric ELISA Kit. To explore the effect of Dex on MSC immunosuppressive ability, MSC were firstly cultured in complete culture medium for 14 d with Dex (10 nmol/L), and then, peripheral blood mononuclear cells (PBMNC) were co-cultured with MSC in 96-well flat-bottom plates for 3 d. Phytohemagglutinin A (PHA, 10 µg/ml) was used to stimulate activation of PBMNC. The concentrations of IFN-γ in culture supernatants was detected by ELISA. The results indicated that there was no obvious difference in representative phenotypes of MSC between experimental and control groups after MSC were treated with low concentration of Dex (10 nmol/L) for 14 d, but the suppression of Dex-treated MSC on lymphocyte activation in same concentration of cells was significantly reduced as compared with control group. After the Dex-treated MSC were co-cultured with IFN-γ for 12 h, the immunoregulatory ability of MSC was recovered in a certain degree. It is concluded that the Dex impairs the immunosuppressive ability of MSC, the IFN-γ can protect and reverse the immunosuppressive ability of MSC impaired by Dex, so that, when the immunoregulatory activity of MSC is investigated, it is necessary to avoid adding Dex in the culture medium.
Cells, Cultured ; Dexamethasone ; adverse effects ; Humans ; Immune Tolerance ; drug effects ; Interferon-gamma ; immunology ; Leukocytes, Mononuclear ; Lymphocyte Activation ; immunology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; immunology

BACKGROUND:As the high proliferation and low apoptosis of the bone marrow in polycythemia vera patients, hematopoietic stem cels transplanted into NOD/SCID mice can differentiate into erythroid cels, but whether hematopoietic stem cels transplantation could improve the hematopoietic function of aplastic anemia mice is not yet reported. OBJECTIVE:To investigate whether transplantation of bone marrow mononuclear cels with JAK2V617F mutation from polycythemia vera patients can influence hematopoietic reconstruction in aplastic anemia mice. METHODS:Severe aplastic anemia mouse models were established by using recombinant human interferon-γplus busulfan, and then, these mouse models were randomly divided into experimental group (n=10) and control group (n=10). Bone marrow mononuclear cels isolated from polycythemia vera patients with positive JAK2V617F mutation were transplanted into the mice in the experimental group via tail vein at 5 days after drug withdrawal.The same volume of normal saline was administered to the control group. Routine peripheral blood test, morphology of bone marrow cels, bone marrow biopsy, and percentage of CD45+ cels in the peripheral blood and marrow were determined at 14 days after transplantation. RESULTS AND CONCLUSION: At 14 days after transplantation, pancytopenia occurred in the experimental group, bone marrow smears showed scattered lymphocytes and hematopoietic progenitors, and bone marrow biopsy presented that hematopoietic tissues were reduced and a smal amount of granulocyte cels and erythroblasts could be seen, but megakaryocytes were rare. In contrast to the control group, there was no improvement in the hematopoietic function of mice in the experimental group. CD45+ cels were detectable in the peripheral blood and bone marrow in the experimental group, but not in the control group; and a higher percentage of CD45+ cels was measured in the bone marrow than in the peripheral blood of experimental group mice. Experimental findings indicate that bone marrow mononuclear cels from polycythemia vera patients with positive JAK2V617F mutation can be engrafted into aplastic anemia mice, but cannot improve the hematopoietic function of mice.

Aim:To investigate the effect of Hemangiopoietin (HAPO) on the hematopoiesis reconstitution in sub-lethally irradiated Balb/c mice.Methods: Balb/c mice were underwent total body irradiation at 700 cGy 137Cs ? radiation and were treated with HAPO or recombinant human granulocyte colony stimulating factor (rhG-CSF) after irradiation. The hematopoiesis reconstitution of mice were detected. Cells from bone marrow of Balb/c mice were cultured with HAPO or rhG-CSF for 24 hours or 72 hours before or after the cells were irradiated. The viability of cells were assessed and the ability of in vitro hematopoiesis reconstitution were also detected. Result: rhG-CSF and HAPO treated mice both showed increased survival rate and increased colony forming units. The peripheral WBC number increased greatly. The HAPO group was most quickest compared with rhG-CSF group and PBS control group. The number of bone marrow cells at day 14 of rhG-CSF group was higher than that in HAPO group, but the number of bone marrow cells at day 32 of rhG-CSF was lower than that in HAPO group. The number of bone marrow cells at day 42 of rhG-CSF was below normal. The number of bone marrow cells at day 42 of HAPO group was nearly normal. The number of CFU-GEMM in HAPO group was most compared with that in rhG-CSF group and PBS control group at day 7, 14 and 21 after radiation. The survival rate of cells after radiation in HAPO group was markedly higher than that in PBS control group, but the survival rate of cells after radiation in rhG-CSF group was no notable difference compared with that in PBS control group. In MTT assay, both HAPO and rhG-CSF incubation stimulated proliferation of bone marrow cell at 72 hours after radiation. Bone marrow cells formed Hematopoietic islands in HAPO group after radiation and were positive for sca-1 and CD31. CD31 positive endothelial cells increased around the Hematopoietic islands. There was no Hematopoietic islands formation, few CD31 positive endothelial cells and no sca-1 positive cells in PBS control group. Conclusion: HAPO can promote hematopoiesis reconstitution in sub-lethally irradiated Balb/c mice. It can increase the survival rate of mice and stimulate the proliferation of hematopoietic stem cells.

Purpose To explore the correlation between the clinicopathological features and postoperative recurrence or metastasis of solid-pseudopapillary neoplasms of the pancreas (SPN).Method The clinicopathological characteristics of 73 SPNs were summarized,the patients' prognosis were followed up by telephone and then the correlation of clinicopathological characteristics and recurrence or metastasis was analyzed.Results 57 female patients and 16 male patients were included in this study.The age range was 7 to 68 years old with the average of 28 and median age of 27.The mean diameter of the tumors was 6.47 cm (range 0.31～ 14 cm).30 cases of tumors were located in the head of pancreas,9 in the body of pancreas,and 33 in the tail of pancreas.One case was a multiple lesion simultaneously located in the body and the tail.All patients were followed up by telephone for mean time 34.8 months with the range of 12 ～ 99 months.Necrosis,calcification,cholesterol crystal,foamy histiocytes,nuclear atypia,pancreatic parenchymal invasion,and perineural invasion had no statistical significance between non-recurrent/metastatic group and recurrent/metastatic group.However,there was significant difference for extra-pancreatic invasion,angiovascular invasion and Ki-67 proliferation index between non-recurrent/metastatic group and recurrent /metastatic group.Conclusion Extra-pancreatic invasion,angiovascular invasion and Ki-67 proliferation index ≥ 4％ have reference significance for predicting recurrence or metastasis of SPN.

OBJECTIVETo investigate the impact of intracellular acidification (IA) on drug resistance of leukemia cells with high P-glycoprotein (P-gp) expression, and to provide a new method for the reversing of multidrug resistance (MDR).

METHODSReal-time PCR was used to determine the expression level of mdr1 gene, and the leukemia cells with high P-gp expression were selected. The specific inhibitor of Na+/H+ exchanger 1 and the "high K+" buffer were used to acidify the cells, and the confocal laser microscopy was used to determine the intracellular pH (pHi) and effect of IA on the accumulation of doxorubicin. The MTT method was used to determine the effect of IA on the cell viability. The flow cytometry was used to detect the effect of IA on the P-gp function, and Western blotting was used to determine the effect of IA on the expression of P-gp.

RESULTSThe pHi was decreased to 7.0, and compared with that of control the mdr1 mRNA expression was decreased to (53.2+/-11.0)% after 1 h, and to (16.6+/-7.0)% after 3 h treatment. The P-gp expression was decreased to (56.0+/-9.0)% of the control after 3 h treatment. The accumulation of Rh123 was 71.03+/-0.47 at pHi 7.0, which was increased obviously as compared to the control group 20.07+/-0.39. The increased accumulation of doxorubicin was also observed by confocal laser microscopy.

CONCLUSIONThe expression and function of P-gp on the patients cells are inhibited by IA.

ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Cell Survival ; drug effects ; Doxorubicin ; pharmacokinetics ; Drug Resistance, Neoplasm ; drug effects ; Guanidines ; pharmacology ; Humans ; Hydrogen-Ion Concentration ; drug effects ; Leukemia ; drug therapy ; metabolism ; RNA, Messenger ; genetics ; Sulfones ; pharmacology ; Tumor Cells, Cultured

This study was aimed to investigate the expression of RHBDD1 gene in patients with chronic myeloid leukemia (CML) and explore its clinical significance. The relative expression levels of RHBDD1 in bone marrow mononuclear cells of healthy controls and CML patients were detected by using real time PCR. The results showed that the expression level of RHBDD1 in CML patients was significantly higher than that in healthy controls. The expression level of RHBDD1 in CML patients with negative BCR/ABL p210 was remarkably higher than that in patients with positive BCR/ABL p210. In patients ≥ 50 years old RHBDD1 expression was lower than the patients < 50 years old. There were no significant relation of RHBDD1 expression with sex of patients. It is concluded that RHBDD1 gene may be involved in the pathogenesis and progression of CML, particularly reflects in the pathogenesis of the patients with negative BCR/ABL p210.
Bone Marrow Cells ; metabolism ; pathology ; Case-Control Studies ; Female ; Gene Expression ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Serine Endopeptidases ; genetics ; metabolism

BACKGROUNDSam68 plays an important role as a multiple functional RNA binding nuclear protein in cell cycle progress, RNA usage, signal transduction, and tyrosine phosphorylation by Src during mitosis. However, its precise impact on these essential cellular functions remains unclear. The purpose of this study is to further elucidate Sam68 functions in RNA metabolism, signal transduction regulation of cell growth and cell proliferation in DT40 cell line.

METHODSBy using gene targeting method, we isolated a mutation form of Sam68 in DT40 cells and described its effect on cell growth process and signal transduction. Southern, Northern, and Western blot, phosphorylation and flow-cytometric analyses were performed to investigate the Sam68 functions.

RESULTSA slower growth rate (2.1 hours growth elongation) and longer S phase (1.7 hours elongation) was observed in the Sam68 mutant cells. Serum depletion resulted in increased amounts of dead cells, and expansion of S phase in mutant cells. Upon B cell cross-linking, the maximal level of tyrosine phosphorylation on BLNK was observed to be significantly lower in mutant cells.

CONCLUSIONSThe proline rich domain of Sam68 is involved in cell growth control by modulating the function of mRNAs in S phase or earlier and the functions as an adaptor molecule in B cell signal transduction pathways.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; physiology ; Animals ; B-Lymphocytes ; cytology ; immunology ; physiology ; Binding Sites ; genetics ; Blotting, Western ; Cell Cycle ; physiology ; Cell Death ; physiology ; Cell Growth Processes ; drug effects ; physiology ; Cell Line, Tumor ; Culture Media, Serum-Free ; pharmacology ; Mutation ; genetics ; Phosphorylation ; Proline ; genetics ; RNA-Binding Proteins ; genetics ; metabolism ; physiology ; Receptors, Antigen, B-Cell ; immunology ; physiology ; Signal Transduction ; drug effects ; physiology ; Tyrosine ; metabolism

The purpose of this study was to investigate the expression feature of a human tumor related gene chp2 in leukemia primary cells and leukemia cell lines, real-time quantitative PCR (RQ-PCR) was performed to detect the expression level of chp2 gene in peripheral blood mononuclear cells from 10 healthy individuals (as control) and 24 cases of leukemia, and in 4 kinds of leukemia cell lines. The results showed that the detection rate of chp2 gene in 10 normal controls was 80%, positive expression was (0.744 +/- 0.682) x 10(5) cps/microl. The expression levels of chp2 mRNA leukemia primary cells and leukemia cell lines were significantly higher than that in the normal control (p < 0.05). The expression levels of chp2 mRNA were higher in AML cells (7 cases), CML cells (6 cases), ALL cells (7 cases) and CLL cells (4 cases), and their expression levels were (11.637 +/- 5.588), (6.122 +/- 3.785), (4.262 +/- 2.561) and (3.434 +/- 1.974) x 10(5) cps/microl respectively. Gene chp2 positively expressed in four kinds of leukemia cell lines, and the expression levels in K562 cells, Jurkat cells, HL-60 cells and M07e cells were (5.243 +/- 1.852), (4.463 +/- 1.621), (4.137 +/- 1.837) and (2.578 +/- 1.137) x 10(6) cps/microl respectively. The expression level in leukemia cell lines was higher than that in primary cells. It is concluded that the human tumor related gene chp2 expression in leukemia primary cells and leukemia cell lines significantly increase, which may play an important role in growth process of leukemia cells.
Calcium-Binding Proteins ; genetics ; metabolism ; HL-60 Cells ; Humans ; Jurkat Cells ; K562 Cells ; Leukemia ; genetics ; metabolism ; pathology ; Neoplasm Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism

5' nucleotides (5'NT), a purine degradative enzyme, is capable of hydrolyzing nucleotide and acting as a phosphotransferase simultaneously. It has critical role in maintaining nucleotide metabolism balance. The present study was aimed to investigate the expression of 5'NT in bone marrow granulocytes (BMGs) from patients with acute myeloid leukemia (AML) and healthy donors comparatively. The BMGs were isolated from bone marrow of 33 patients with AML and 6 healthy donors by using lymphocyte isolating solution. The reactivity of 5'NT was detected by electron microscope and cytochemistry of cytidine monophosphate (CMP). The positive BMG ratio and their index were calculated on the base of ultrastructural observation semiquantitatively. The results indicated that electron microscopy revealed plasma membrane reacting pattern of CMP. Most BMGs from normal donors were CMP negative or exhibited lower active degree. All cases of M(0), M(1), M(2) and t (8; 21) showed high positive percentages and high indexes of BMGs, but no statistic differences between them. APL of t (15; 17) shared lower percentages and indexes than other subtypes. There was no significant difference between APL and normal donors statistically. In conclusions, the results suggested the expression of 5'NT may be associated with BMG differentiation in AML, and APL of t (15; 17) may be a highly differentiated leukemia subtype.
5'-Nucleotidase ; metabolism ; ultrastructure ; Adolescent ; Adult ; Aged ; Bone Marrow Cells ; cytology ; enzymology ; Child ; Female ; Granulocytes ; enzymology ; Humans ; Leukemia, Myeloid, Acute ; classification ; enzymology ; Male ; Middle Aged ; Young Adult

OBJECTIVETo investigate the feasibility and benefits of co-culture of cryopreserved islets with small intestinal submucosa (SIS).

METHODSPurified rat islets cryopreserved for one month were divided into SIS group and control group, and after culture in standard islet culture media RPMI1640 for 1 week, the morphology and function of the islets were assessed.

RESULTSThe SIS protects the fragile islets from damage by cryopreservation, and increased the recovery from (60.6-/+3.3)% to (91.7-/+1.8) % (P<0.05). Compared with the control group, incubation of the islets of the SIS group in high-glucose (16.7 mmol/L) solution resulted in significantly enhanced insulin secretion (23.7-/+1.6 vs 12.5-/+1.1 mU/L, P<0.05). When the islets were incubated in high-glucose solution containing theophylline, the calculated stimulation index of SIS group was about 3-fold higher than that of the control group.

CONCLUSIONCo-culture of cryopreserved rat islets with SIS can increase the recovery of islet cells and improve their function.

Animals ; Coculture Techniques ; Cryopreservation ; methods ; Glucose ; pharmacology ; Insulin ; secretion ; Intestinal Mucosa ; cytology ; drug effects ; physiology ; Intestine, Small ; cytology ; drug effects ; physiology ; Islets of Langerhans ; cytology ; drug effects ; physiology ; Male ; Rats ; Rats, Wistar ; Theophylline ; pharmacology