Animals ; Gene Expression Profiling ; Gene Silencing ; Hematologic Neoplasms ; genetics ; metabolism ; Hematopoiesis ; genetics ; Humans ; MicroRNAs ; metabolism ; physiology ; Neoplasms ; genetics ; metabolism ; Oligonucleotide Array Sequence Analysis ; T-Lymphocytes ; metabolism

Cell-Derived Microparticles ; Disease Progression ; Humans ; Leukemia ; pathology

In order to investigate the molecular mechanisms of SARI expression regulation in chronic myeloid leukemia (CML), 46 patients with CML and 40 healthy volunteers were recruited in this study. SARI expression in the peripheral blood mononuclear cells (PBMNC) of CML patients and healthy volunteers was assayed by using real-time quantitative PCR. K562 cells were in vitro incubated with the BCR-ABL inhibitor STI571 (imatinib) at 37°C and 5% CO2 for 24 hours, then SARI expression was detected by using real-time quantitative PCR. All experiments were repeated three times. The results showed that as compared with healthy volunteers, the expression of SARI mRNA in PBMNC of CML patients presented a lower level (p < 0.001). After exposure of K562 cells to STI571 (2.5 µmol/L) for 24 hours, the SARI expression was higher than that in K562 cells treated without STI571 (p < 0.001). It is concluded that the suppression of SARI expression is involved in CML pathogenesis, and BCR-ABL mediates the down-regulation of SARI mRNA expression in K562 cells. These findings suggest a new orientation for gene therapy in CML patients.
Basic-Leucine Zipper Transcription Factors ; genetics ; Benzamides ; Fusion Proteins, bcr-abl ; antagonists & inhibitors ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; Imatinib Mesylate ; K562 Cells ; Piperazines ; pharmacology ; Pyrimidines ; pharmacology ; Tumor Suppressor Proteins ; genetics

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.
Adolescent ; Adult ; CD4 Antigens ; analysis ; Female ; Humans ; Immune Tolerance ; immunology ; Interleukin-2 Receptor alpha Subunit ; analysis ; Lymphoma, B-Cell ; blood ; immunology ; Male ; T-Lymphocyte Subsets ; immunology ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo investigate the expression and significance of survivin protein and mRNA in cord blood (CB) CD(34)(+) stem/progenitor cells.

METHODSSurvivin level in CB CD(34)(+) cells was assessed by immuno-histochemistry and reverse transcription polymerase chain reaction (RT-PCR).

RESULTSSurvivin expressed in fresh CB CD(34)(+) cells, its protein level was 9.4 +/- 1.2, faint signal of the mRNA transcript band was detected. In the in vitro culture of the cells, hematopoietic growth factors could upregulate survivin expression, especially the combination of SCF, FL and Tpo. After 3 days culture, its protein level was 25.2 +/- 5.3, and mRNA transcript increased significantly. Survivin expression dropped down 24 hours after withdraw of these cytokines.

CONCLUSIONSurvivin is not a specific anti-apoptotic protein of cancer, and also expressed in normal hematopoietic stem/progenitor cells. It plays important regulating roles in the process of hematopoiesis.

Antigens, CD34 ; blood ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Cell Growth Factors ; pharmacology ; Hematopoietic Stem Cells ; drug effects ; metabolism ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; physiology ; Neoplasm Proteins ; biosynthesis ; genetics ; physiology ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

A variety of immuno-deficient animal models has been used in the detection of human hematopoietic stem/progenitor cells and up to date make many remarkable contribution to the study of detectron cell biology. An ideal immuno-deficient animal should not only have complete immune defects, be able to transplant human hematopoietic stem/progenitor cells with high efficacy, but also survive long enough for observation. In this review, the development of immuno-deficient mice from nude, SCID, to NOD/SCID etc is introduced and much attention is put on the features, priority and shortcomings of those key animal models and their application in HSC research. The progress, the future directions and prospects of these immuno-deficient mice models are stressed.
Animals ; Hematopoietic Stem Cell Transplantation ; methods ; Hematopoietic Stem Cells ; cytology ; immunology ; metabolism ; Humans ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Models, Animal ; Transplantation, Heterologous

OBJECTIVETo investigate the rapid construction of enhanced green fluorescent protein (EGFP) labeled recombinant adenovirus containing hVEGF165 and its distribution and efficiency in bone marrow transplanted mice.

METHODSThe recombinant adenovirus Ad-EGFP/hVEGF165 was rapidly constructed by using AdEasy system based on the homologous recombination in bacteria, and its property was studied in vitro. Then 3x10(8) PFU adenovirus was injected into BALB/c mice via the tail vein accepted syngeneic bone marrow transplantation. The in vivo distribution of adenovirus and plasma levels of VEGF were measured at different phases.

RESULTSThe adenovirus Ad-EGFP/hVEGF165 was quickly constructed by homologous recombination in bacteria using AdEasy system. The purified particles were homogenous hexagon with titers between 10(10) PFU/ml and 10(11) PFU/ml. The Hela cells infected with Ad-EGFP/hVEGF165 did not show cytopathic effects after several times passages. Under the fluorescent microscope, EGFP was revealed in the heart, lung, liver, spleen, kidney and intestine of mice at different phases. RT-PCR and immunohistochemistry showed hVEGF165 expressed significantly. No obvious damages were observed in different organs by HE staining. The plasma level of hVEGF165 was up to 866.67+/-97.13 pg/ml.

CONCLUSIONThese results suggested that the construction of adenovirus vector by homologous recombination in bacteria was an efficient and time-saving method, and high titer adenovirus could successfully mediate the safe and stable expression of hVEGF165 in post bone marrow transplanted mice. All these would make further gene therapy in bone marrow transplantation possible.

Adenoviridae ; genetics ; Animals ; Bone Marrow Transplantation ; Female ; Gene Transfer Techniques ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Recombination, Genetic ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

To investigate the expression and function of the receptors of early-acting cytokines on cord blood CD34(+) hematopoietic stem/progenitor cells, the studies of Flt3 and c-kit were undertaken at the gene and protein levels. Fresh and cultured cord blood CD34(+) stem/progenitor cells were analyzed by flow cytometric two-color direct immunofluorescence methods and RT-PCR, while function of receptors was studied in vitro. It was found that (68.8 +/- 15.4)% of CD34(+) cells expressed Flt3, (50.6 +/- 12.7)% of CD34(+) cells expressed c-kit, the proportion of CD34(+) cells expressing Flt3 and c-kit decreased as in vitro culture time extended. It was concluded that cord blood CD34(+) stem/progenitor cells are capable of expressing Flt3 and c-kit for as long as 2 - 3 weeks in liquid medium, during the first week of culture, SCF and FL enhanced the generation of cells and progenitors notably.
Antigens, CD34 ; blood ; Cells, Cultured ; Fetal Blood ; cytology ; Flow Cytometry ; Hematopoietic Stem Cells ; chemistry ; Humans ; Proto-Oncogene Proteins ; analysis ; genetics ; physiology ; Proto-Oncogene Proteins c-kit ; analysis ; genetics ; physiology ; RNA, Messenger ; analysis ; Receptor Protein-Tyrosine Kinases ; analysis ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cell Factor ; pharmacology ; fms-Like Tyrosine Kinase 3

To evaluate expression of caspase-3 protein in CD34(+) cells from cord blood (CB) and its significance during culture in vitro with various growth factors, RT-PCR, Western blot and flow cytometry were used to determine cell proliferation, apoptosis and expression of caspase-3 in CD34(+) cells during culture in vitro. The results demonstrated that caspase-3 mRNA was constitutively expressed at a low level in freshly isolated CD34(+) cells. Expression of caspase-3 mRNA and protein was upregulated when these cells were expanded in suspension culture with growth factors for 3 days. However, only the 32 kD inactive caspase-3 proenzyme was detected in the freshly isolated CD34(+) cells and those cells after 3 days expansion with cytokines. Along with the culture time extension (7 days) in vitro, especially without early-effective cytokines SCF or FL existence, caspase-3 was activated and a cleavage 20 kD was detected. It is concluded that the caspase-3 is involved in apoptosis of primitive CD34(+) cells during expansion in vitro, and early-effective cytokines, SCF and FL, could conserve hematopoietic stem cells and suppress apoptosis.
Antigens, CD34 ; blood ; Apoptosis ; Caspase 3 ; Caspases ; analysis ; physiology ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; enzymology ; physiology ; Humans ; Infant, Newborn ; Reverse Transcriptase Polymerase Chain Reaction

Objective To find out pathologieal change and the expression of neuron specific enolage (NSE)in the hippocampus dentate gyrus granular cell layer of epilepsy patients,to investigate the relationship between the pathological change and the cause of the epilepsy.Methods The specimens of hippocampus were from 9 epilepsy patients and 20 normal persons and the pathological change were investigated under the staining of the hematoxylin and eosin staining.The expression of NSE in the hippocampns denmte gyrus granular cell layer of epilepsy patients was detected by immunohistochemistry(IHC)with specific antibody,and the rate of NSE positive nenrons was evaluated.Results The nuclear pyknosis was observed in all of hippocampus from the epilepsy patients and some neurons were swelling.The positive of NSE was showed to have yellow granule;the rate of NSE positive was 28.66%.The expression of NSE of the neurons in the hippocampus dentate gyrus granular cell layer was.significantly reduced in epilepsy patients(7.9±5.6)%.The normal neuron nuclear was big and round in the middle of the cell and the nucleolus could be seen easily.The expression of NSE of the neurons in the hippocampus dentate gyrus granular cell layer was(39.0±17.4)%.Compared with normal group,the number of neurons with yellow granule was reduced.The difference of the NSE expression rate between the two group was statistically significant(t＝-5.13,P＜0.01).Conclusions The result suggests that the pathological abnormality and the reduced expression of NSE on the hippocampus dentate gyrus granular cell layer neurons could be one of the main reasons of the occurring of epilepsy.