To study the methylation in the promoter of LRP15 gene and its relationship with gene expression and to explore the possible mechanism of regulating LRP15 gene methylation, the methylation in the promoter of LRP15 gene in K562 cell line was detected by MS-PCR. Then K562 was exposed to 5-aza-2'-deoxycytidine (CdR) and trichostatin (TSA), to determine whether the silencing of LRP15 gene by de novo methylation could be reversed. As a result, it was confirmed by MS-PCR that the promoter of LRP15 was hypermathylated in K562 cell line, and lost its transcription activity. After CdR, with or without TSA, the silencing of LRP15 gene by de novo methylation can be reversed. Observation demonstrated that the expression of LRP15 was controlled by methylation in its promoter in K562. It is suggested that methyltransferase inhibitor and deacetylase inhibitor may be effective agents in leukemia therapy.
Azacitidine ; analogs & derivatives ; pharmacology ; DNA Methylation ; DNA Modification Methylases ; antagonists & inhibitors ; Enzyme Inhibitors ; pharmacology ; Gene Expression Regulation ; drug effects ; Histone Deacetylase Inhibitors ; Humans ; Hydroxamic Acids ; pharmacology ; K562 Cells ; Neoplasm Proteins ; biosynthesis ; genetics ; Polymerase Chain Reaction ; methods ; Promoter Regions, Genetic ; genetics

To investigate the effect of GFP fused to C terminal of integrin alpha(IIb) on the biosynthesis and expression of alpha(IIb) beta(3) compound, the alpha(IIb) GFP expression plamid, named palpha(IIb) GFP, the cDNA of alpha(IIb) was constructed from p3.1-2b and fused to pEGFP-N1 in frame. When the sequence of palpha(IIb) GFP was confirmed by sequencing it was transferred to Chinese Hamster Ovary (CHO) cells with or without p3.1-3a expressing integrin beta(3). Then the expression of alpha(IIb) GFP fusion protein was confirmed by Western blot and then its subcellular localization was determined with laser confocal scanning microscopy. The results showed that the target gene was cloned into recombinant vector by restriction analysis and sequencing. Overexpression of the fusion protein in the transfected CHO cells was identified with Western blot. Subcellular localization analysis confirmed that alpha(IIb) GFP was expressed in CHO cells and could be transferred from endoplasmic reticulum to Golgi apparatus. It is concluded that the eukaryotic expression plasmid containing alpha(IIb) GFP fusion gene is successfully constructed. GFP fused to the cytoplasmic tail of integrin alpha(IIb) allows the normal expression of alpha(IIb) beta(3) in CHO cells.
Animals ; Blotting, Western ; CHO Cells ; Cricetinae ; Cricetulus ; Endoplasmic Reticulum ; metabolism ; Golgi Apparatus ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Microscopy, Confocal ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection

To investigate the function and molecular mechanism of p21(WAF1/Cip-1) expression in MOLT-4 cells induced by HDAC inhibitor TSA, the expression pattern of p21(WAF1/Cip-1) and the distribution of cell cycle in TSA treated cells were analyzed. The results showed that TSA could effectively induce G(2)/M arrest and apoptosis of MOLT-4 cells. Kinetic experiments demonstrated that p21(WAF1/Cip-1) were upregulated quickly before cell arrested in G(2)/M and began decreasing at the early stage of apoptosis. Meanwhile, the proteasome inhibitor MG-132 could inhibit the decrease of p21(WAF1/Cip-1) at the early stage of apoptosis, which showed that proteasome pathway involved in p21(WAF1/Cip-1) degradation during the TSA induced G(2)/M arrest and apoptosis responses. This study also identified that the protein level of p21(WAF1/Cip-1) was highly associated with the cell cycle change induced by TSA. Compared to cells treated by TSA only, exposure MOLT-4 cells to TSA meanwhile treatment with MG-132 increased the protein level of p21(WAF1/Cip-1) and increased the numbers of cell in G(2)/M-phase, whereas the cell apoptosis were delayed. It is concluded that p21(WAF1/Cip-1) plays a significant role in G(2)/M arrest and apoptosis signaling induced by TSA in MOLT-4 cells.
Apoptosis ; drug effects ; Blotting, Western ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; Enzyme Inhibitors ; pharmacology ; Flow Cytometry ; Histone Deacetylase Inhibitors ; Humans ; Hydroxamic Acids ; pharmacology ; Leukemia, Myeloid ; metabolism ; pathology

One of the important approaches for further prolonging remission duration and eradicating minimal residual disease in acute leukemia is immunotherapy. Four kinds of immunotherapy for acute leukemia are under investigation: (1) monoclonal antibodies, among them, Mylotarg (cytotoxic antibiotic calicheamicin linked to CD33 Mab) is given for the treatment of refractory or relapsed acute myeloid leukemia and molecular relapse in acute promyelocytic leukemia with good results, Campath-1H (antiCD52 Mab) is administered in the treatment of prolymphocytic leukemia and Rituximab (anti-CD20 Mab) in B-PLL with high complete remission rates. Other Mabs under preclinical and clinical trials include anti-IL-2 receptor Mab for the treatment of acute T lymphocytic leukemia, anti-220 kD Mab-6G7 for acute leukemias, recombinant immune toxin BL22 (anti-CD22) for hairy cell leukemia and Mabs labeled with radio-isotopes for different types of acute leukemias; (2) adoptive cellular immunotherapy using cytokine-induced killer cell, alloreactive NK cells, allogeneic or autologous leukemic-specific CD8(+) cytotoxic T lymphocytes, and other immune effector cells; (3) cytokines and other immune modulators comprising IL-2, IL-12, GM-CSF, CD40L, FLT-3L and thalidomide and its derivatives; (4) leukemia vaccines of several different formulations including antigen-specific, leukemia cell-based, leukemia antigen-pulsed dendritic cell (DC) and leukemia-derived DC vaccines, the latter two formulations are more attractive. In conclusion, up to now, the most effective example of immunotherapy in acute leukemia is provided by the administration of Mabs, and the majority of other approaches in immunotherapy for acute leukemia although promising, need further studies.
Acute Disease ; Adoptive Transfer ; methods ; Antibodies, Monoclonal ; immunology ; therapeutic use ; Cancer Vaccines ; therapeutic use ; Humans ; Immunotherapy ; methods ; trends ; Leukemia ; immunology ; therapy

During mammalian ontogeny, hematopoietic activity can be found in distinct anatomical sites, which con-tribute to primitive or definite hematopoiesis. The origin of the hematopoietic stem cell (HSC) has been a controversial issue in the field of hematopoiesis. It has long been believed that the origin derives from the extra-embryonic yolk sac. However, there is now considerable evidence that the first adult repopulating HSC is autonomously generated from a distinct region within the embryonic mesoderm, the aorta-gonad-mesonephros (AGM) region. This review describes the origin and precise location of HSC in the embryo and in AGM region, the hematopoietic microenvironment and the hematopoietic regulatory mechanisms in AGM region.
Animals ; Aorta ; cytology ; embryology ; Gonads ; cytology ; embryology ; Hematopoiesis ; physiology ; Hematopoietic Stem Cells ; cytology ; physiology ; Hematopoietic System ; cytology ; embryology ; Humans ; Mesonephros ; cytology ; embryology

There has been an increasing interest in recent years on mesenchymal stem cell (MSC). It is well known that MSCs are capable of self-renewal and differentiating into many cell lineages. MSC can be expended to a large quantity that is required for clinical transplantation. Recent studies show that MSC have potential application in immune diseases due to their unique immunologic characteristics, such as low immunogenicity and immunoregulatory function. But their immunoregulatory mechanism is not yet clear. This review discusses the advances in researches on the mechanism of MSCs' immunoregulatory function and potential clinical application in immune disease and organ transplantation.
Animals ; B-Lymphocytes ; immunology ; Graft vs Host Disease ; immunology ; Humans ; Immune Tolerance ; immunology ; Immunotherapy ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; cytology ; immunology ; T-Lymphocytes ; immunology

To develop a real-time FQ-PCR method for quantifying human ermap, a set of primers and a fluorescent probe were designed by primer express 2.0. pBluescriptSK(+) plasmid contained ermap cDNA was transcribed to generate calibration standards for quantification. A real time FQ-PCR method was established. The results showed that when the concentrations of DNA to be amplified were ranged from 1.725 x 10(7) to 1.725 x 10(10) cps/ml, there was a good correlation between template concentration and cycle threshold, and the correlation coefficient reached to -0.999376. In conclusion, real time FQ-PCR which is specific, sensitive and accurate can be used to further research on human ermap.
Blood Group Antigens ; genetics ; Butyrophilins ; DNA, Complementary ; chemistry ; genetics ; Fluorescent Dyes ; chemistry ; Fluorometry ; methods ; Humans ; Polymerase Chain Reaction ; methods ; Reproducibility of Results

To evaluate the clinical efficacy of recombinant human Interleukin 11 in the treatment of pre-aplastic anemia, six patients with pre-aplastic anemia were injected with rhIL-11 of 6 million units once a day during 7-14 days. Blood platelet counts were taken on day 8, 15, 30 and 60 after the treatment, and bone marrow examination was performed on day 15 as compared with those before treatment. The results showed that platelet counts in 3 out of 6 patients increased remarkably (50%), one of the six increased moderately (16.7%), another case of the six increased slightly (16.7%), platelet in one out of six did not significantly increase (16.7%), the total efficacy rate is 83.3%, the amount of megakaryocyte in bone marrow of all six patients increased, the side effect of the rhIL-11 treatment was light. In conclusion, the efficacy of recombinant human Interleukin-11 in the treatment of thrombocytopenia patients with pre-aplastic anemia is satisfactory. As the number of the cases is too small to conclude, further exploration needs accumulation of more applications.
Adult ; Aged ; Anemia, Aplastic ; drug therapy ; pathology ; Chronic Disease ; Female ; Humans ; Interleukin-11 ; genetics ; therapeutic use ; Male ; Middle Aged ; Platelet Count ; Recombinant Proteins ; therapeutic use ; Thrombocytopenia ; blood ; drug therapy ; Treatment Outcome

UNLABELLEDGlucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy. To date, about 126 mutations in the G6PD gene have been detected, among which 17 mutations were found in Chinese. The most common mutations are: 1376 G-->T and 1388 G-->A, both in exon 12; 95 A-->G in exon 2, which amounted to more than 50% of mutations representing various regions and ethnic groups in China. A large-scale screening and genotypic analysis was held in Shui people in Sandu of Guizhou. To investigate the incidence and the molecular basis of G6PD deficiency of Guizhou Shui people, NBT qualitative and G6PD/6PGD quantitative methods were used to detect G6PD deficiency in 1,090 Shui people from the general people belonging to Sandu of Guizhou. By means of mis-matched primers amplified the G6PD gene, the products were 234 bp, 280 bp and 345 bp in length, then restriction enzyme analysis was used to detect the most common Chinese G6PD mutations, 1376 G-->T, 1388 G-->A and 95 A-->G. The results showed that out of the 1,090 samples, 98 G6PD deficiency samples were found. The incidence of G6PD deficiency was 8.99%. 24 cases of 1376 G-->T, 12 cases of 1388 G-->A, 9 cases of 95 A-->G were detected. A sample with 1376 G-->T and 95 A-->G mutation was found in a girl. It was reported for the first time.

IN CONCLUSION1376 G-->T, 1388 G-->A, 95 A-->G mutations are the common G6PD mutations in Shui people in Sandu of Guizhou. The results indicates that different national minorities of Chinese may originated from a common ancestor.

China ; epidemiology ; Female ; Gene Frequency ; Genotype ; Glucosephosphate Dehydrogenase ; genetics ; Glucosephosphate Dehydrogenase Deficiency ; enzymology ; epidemiology ; genetics ; Humans ; Incidence ; Male ; Point Mutation

The study was aimed to establish a standard procedure for human umbilical cord blood bank. The hematopoietic nucleated cells in cord blood were processed by using sedimentation and centrifugation method. After finishing CD34(+) cell counting, hematopoietic progenitor cell assay, microbial culture, infectious disease test and HLA typing, cord blood units were stored in the liquid nitrogen for further application. The results showed that nucleated cells of cord blood were (10.94 +/- 2.74) x 10(8) per unit; recovery rate of nucleated cells was (79.82 +/- 17.76)%. CD34(+) cells in cord blood were counted as (51.62 +/- 30.53) x 10(5) per unit. Eight units of cord blood were thawed after two years of cryopreservation, the recovery rate of nucleated cells, CD34(+) cells and CFU-GM were (91.4 +/- 6.0)%, (84.6 +/- 20.0)% and (85.8 +/- 14.9)% respectively. It is suggested that the methods and procedure reported for processing and cryopreservation of hematopoietic stem/progenitor cells in the human umbilical cord blood is effective.
Antigens, CD34 ; blood ; Blood Preservation ; methods ; Cell Separation ; methods ; Colony-Forming Units Assay ; Cryopreservation ; methods ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; immunology ; Humans