The object of this study was to explore the effects of Panax notoginosides (PNS) on proliferation and differentiation of human CD34(+) stem/progenitor cells. CD34(+) cells were isolated from human bone marrow by using immune beads of Dynal M- 450 system. The cells were exposed to PNS at different concentrations in both liquid and semi-solid culture for 14 days. The cells were marked with monoclonal antibodies and analyzed by flow cytometry after culture. The CFU-Mix colony formation from CD34(+) cells was assayed. The results showed that: (1) The yield of CD34(+) cells after being selected by immune beads were (1.03 +/- 0.74)% out of bone marrow nuclear cells with purity of 86% - 93%. (2) PNS (10 - 25 mg/L) stimulated the proliferation of CD34(+) cells, and raised the colony numbers of CFU-Mix obviously in vitro. PNS 25 mg/L was the optimal concentration to promote proliferation of CD34(+) cells, the increasing rate of CFU-Mix colony was (34.7 +/- 16.0)%. (3) The differentiation of CD34(+) cells was induced by exposure to PNS (25, 50 and 100 mg/L) in liquid culture for 14 days. The percentages of CD33(+) and CD15(+) cells were increased after PNS exposure, which were significantly higher than those of control (P < 0.01), however CD71(+) and G-A(+) cells were no obviously difference after PNS treatment. In conclusion, Panax notoginosides not only promote the proliferation of CD34(+) cells, but also induce the differentiation committed to granulocytes.
Antigens, CD ; analysis ; Antigens, CD34 ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Cell Differentiation ; drug effects ; Cell Division ; drug effects ; Ginsenosides ; pharmacology ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Lewis X Antigen ; analysis ; Sialic Acid Binding Ig-like Lectin 3

A little is known about the specific marker on the surface of acute leukemia cells, leading to the lack of the specific diagnosis method for acute leukemia. Therefore, in this study, cell-systematic evolution of ligands by exponential enrichment (cSELEX) was performed to screen the aptamers binding to CD33(+)/CD34(+) cells from the patients with acute myeloblastic leukemia (AML) of M(2) subtype (AML-M₂) so as to provide the basis for finding the specific marker on the surface of AML-M(2) CD33(+)/CD34(+) cells. Firstly, AML-M₂ CD33(+)/CD34(+) cells were sorted and used as targeted cells, and normal CD33(+)/CD34(+)cells were used as counter-targeted cells; the aptamers binding to CD33(+)/CD34(+) cells from patients with AML-M₂ were screened from the single strand deoxyribonucleic acid (ssDNA) library by cSELEX. Subsequently, each aptamer structure was analyzed after cloning and sequencing. The results indicated that after 13 round of screenings, the enrichment of aptamers in the ssDNA library was ranged from 0.7% to 52.9%, and reached steady state at 13th round screening. Sequence analysis for 30 aptamers showed that most of the aptamers born one of the three conserved sequences of CCCCT, CTCTC, and CTCAC. Secondary structure analysis indicated that three different secondary structures existed in these aptamers. It is concluded that the aptamers binding to the AML-M(2) CD33(+)/CD34(+) cells are successfully screened, which lay the basis for further looking for the specific marker on the surface of AML-M₂ CD33(+)/CD34(+) cells, and the molecular diagnosis of the AML-M₂ leukemia.
Antigens, CD ; genetics ; immunology ; Antigens, CD34 ; genetics ; immunology ; Antigens, Differentiation, Myelomonocytic ; genetics ; immunology ; Aptamers, Nucleotide ; metabolism ; Biomarkers ; Flow Cytometry ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; genetics ; immunology ; Nucleic Acid Conformation ; SELEX Aptamer Technique ; Sialic Acid Binding Ig-like Lectin 3

OBJECTIVEGene therapy of leukemia is a new and effective method. It is known to all that the pathogenesis and development of leukemia are related to a variety of genes. Survivin is a member of inhibitors of apoptotic proteins (IAP). Its cDNA was cloned from target cell protease receptor-1 (EPR-1). It is expressed in common tumors, but there is no expression in normal and mature tissues. High expression of survivin was detected in leukemic cells. The present study was conducted to examine the role of survivin in the differentiation of leukemic cells by using antisense-oligonucleotides.

METHODSHuman leukemic cell K562 was used as the model for the study. K562 cells were divided into 4 groups randomly: antisense oligonucleuotide (ASON) group, nonsense oligonucleotide (NSON) group, lipofectin group and control group. There were 5 samples in each group, and the experiment was repeated for three times. ASON was designed with the reference to targeting survivin mRNA. K562 cells were cultured in RPMI1640 contained fetal cattle serum at a concentration of about 10 percent. Cell transfection was induced by lipofectin. Forty-eight hours after thansfection, the morphology and ultrastructure were observed. Twenty-four hours and 48 hours after thansfection, the function of K562 cells was detected by benzidine staining, POX staining and NBT staining, respectively. The mean fluorescence intensity of CD33 was determined by flow cytometry. The method of immunohistochemistry was used to examine the protein level of survivin.

RESULTSAfter thansfection with ASON, the size of K562 cells was reduced, but the cytoplasm was increased. The metarubricyte, segment granulocyte, apoptotic cells could be found. Morphologically and ultrastructurally, erythroid and myelocytic differentiation was observed. The positive level of benzidine staining in ASON group (11.90 +/- 2.30 at 24 h and 18.20 +/- 2.93 at 48 h) was higher than that of NSON group, lipofectin group and control group, respectively. The positive level of POX staining in ASON group (17.40 +/- 3.54 at 24 h and 29.40 +/- 3.70 at 48 h) was also higher than that of any other groups. The positive level of NBT staining in ASON group (7.50 +/- 2.26 at 24 h and 12.10 +/- 2.63 at 48 h) was significantly higher than that of NSON group, lipofectin group and control group, respectively (P < 0.01). In ASON group, the mean fluorescence intensity of CD33 (21.43 +/- 1.61 at 24 h and 14.86 +/- 1.20 at 48 h) was significantly lower than that of any other groups (P < 0.01). After thansfection for 24 h, the protein level of survivin in ASON group was decreased significantly compared to that of control group. There was no difference in survivin protein level amongst ASON group, NSON group and lipofectin group at 24 h (P > 0.05). Forty-eight hours after thansfection, the protein level of survivin was decreased significantly.

CONCLUSIONSASON targeting survivin can induce K562 to erythroid and myelocytic differentiation. Survivin is related to differentiation of K562 cells, and it can be a target of gene therapy for leukemia.

Antigens, CD ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Cell Differentiation ; Humans ; Inhibitor of Apoptosis Proteins ; K562 Cells ; Microtubule-Associated Proteins ; analysis ; antagonists & inhibitors ; genetics ; physiology ; Oligonucleotides, Antisense ; genetics ; Sialic Acid Binding Ig-like Lectin 3 ; Transfection

The phenotypes of the bone marrow cells in various subtypes of myelodysplastic syndromes (MDS) and its clinical implication were explored. The antigen expression of a panel of antigens expressed in marrow cells from 30 patients with subtypes of MDS was assayed by alkaline phosphatase anti-alkaline phosphatase method. The results showed that the expression of myeloid antigens appeared abnormality, CD13 and CD33, found on granulocyte and macrophage precursors, increased, and CD15 decreased. There were no significant changes for monocytic antigen CD14 and lymphoid antigens CD7 and CD10. CD34 was increased in RAEB/RAEB-t and was not increased in RA/RAS patients. CD71, expressed by erythroblast and proliferative cells, was higher in all subtypes of MDS than that in control group. It is suggested that the bone marrow cells from MDS patients showed abnormality of more than two series of immunophenotypes, detection of immunophenotype in MDS cells might be contributed to the diagnosis and predicting prognosis.
Adult ; Aged ; Antigens, CD ; analysis ; Antigens, CD34 ; analysis ; Antigens, CD7 ; analysis ; Antigens, Differentiation, B-Lymphocyte ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Bone Marrow Cells ; immunology ; CD13 Antigens ; analysis ; Female ; Humans ; Immunophenotyping ; Lewis X Antigen ; analysis ; Lipopolysaccharide Receptors ; analysis ; Male ; Middle Aged ; Myelodysplastic Syndromes ; immunology ; pathology ; Neprilysin ; analysis ; Receptors, Transferrin ; Sialic Acid Binding Ig-like Lectin 3

To study the effect of interleukin-15 (IL-15) on the proliferation, differentiation and apoptosis of MDS CD34(+) cells, CD34(+) cells of high enrichment were separated by MACS system, and cultured in liquid media with different concentration of IL-15 in treated group and without IL-15 in the control group. Apoptosis of hematopoietic precursors was assayed by propidium iodine staining and cell by FCM, and the other MDS CD34(+) cells were stained by cytochemical staining after culture. The results showed that after culture with IL-15 the proliferation and differentiation of MDS CD34(+) cells were obviously promoted. It was found the every lineage of mature cells developed, the expressions of cell surface antigens CD71, CD33 and CD19 all increased in the MDS CD34(+) cell treated with IL-15. It is suggested that IL-15 stimulates the proliferation and differentiation of MDS CD34(+) cells, and partly shows anti-apoptosis effects which may be applicable to the therapy MDS.
Antigens, CD ; immunology ; Antigens, CD19 ; immunology ; Antigens, CD34 ; immunology ; Antigens, Differentiation, Myelomonocytic ; immunology ; Apoptosis ; drug effects ; Bone Marrow Cells ; drug effects ; immunology ; pathology ; Cell Cycle ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Flow Cytometry ; Humans ; Interleukin-15 ; pharmacology ; Microscopy, Fluorescence ; Myelodysplastic Syndromes ; blood ; immunology ; pathology ; Receptors, Transferrin ; immunology ; Sialic Acid Binding Ig-like Lectin 3

The purpose of this study was to investigate the immunophenotyping characteristics of adult acute lymphoblastic leukemia (ALL) patients in groups of different ages. Immunophenotyping was performed in 260 ALL patients by flow cytometry using a panel of monoclonal antibodies and CD45/SSC gating. The results indicated that (1) all the 82 cases of T-cell acute lymphoblastic leukemia (T-ALL) expressed CD7 (100%) while the positive rate of CD2 remarkably decreased with aging. The positive rate of CD2 in patients aged 14 to 18 years (adolescents) was 91.67%, which is significantly higher than that in cases aged 19 to 35 years (young adults) and > 35 years (older adults) (65.71% and 43.48% respectively, p < 0.05); the positive rate of CD34 and HLA-DR increased with aging, there was significant difference of the HLA-DR expression between the older adults group (39.13%) and the other two groups (4.17% in adolescents and 11.43% in young adults respectively (p < 0.05). Moreover, there were significant differences of the myeloid antigen (MyAg) and CD13 expression between the older adults and younger adults (p < 0.05). (2) As to adult B-cell acute lymphoblastic leukemia (B-ALL), the positive rates of CD19 and HLA-DR in 178 cases were 100%; the positive rate of CD33 in young adults was significant higher than that in adolescents (p < 0.05), the differences of the other marker expressions failed to reach statistical significance in adult B-ALL patients. It is concluded that the immunophenotypes of adult T-ALL are evidently heterogeneous in different ages, and expression with more aberrant phenotypes indicates poor prognostic significance in patients older than 35 years. There is no significant association of immunophenotypes with ages among different age groups of adult B-ALL.
Adolescent ; Adult ; Age Factors ; Aged ; Antigens, CD ; immunology ; Antigens, CD19 ; immunology ; Antigens, CD34 ; immunology ; Antigens, Differentiation, Myelomonocytic ; immunology ; CD13 Antigens ; immunology ; CD2 Antigens ; immunology ; Female ; Humans ; Immunophenotyping ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; immunology ; Sialic Acid Binding Ig-like Lectin 3 ; Young Adult

The study was aimed to investigate the immunological characteristics and prognosis of acute myeloid leukemia (AML) M(1) and to find the main points in immunology to differentiate AML M(1) from M(2), and M(1) from ALL (proB, preB, T). Immunophenotyping was performed in 41 AML M(1) patients by three-color flow cytometry analysis using CD45/SSC gating, meanwhile the cytogenetic analysis was performed in 17 patients. 51 newly diagnosed AML M(2) patients and 58 newly diagnosed ALL patients were used as control at the same time. The results showed that the positive rate of CD33 in M(1) was 100%, which was high in sensitivity, but low in specificity; the positive rate of CD11b, CD15, MPO, CD117 in M(1) were significantly lower than that in M(2) (p < 0.05); the positive rate of T-lineage antigen in Ly + AML M(1) was higher than that in M(2) (p < 0.05); compared with ALL ProB, M(1) had high expression of HLA-DR, simultaneously myeloid antigen CD13, CD15, CD33, CD117, MPO and T-lineage antigen CD4, CD7 were all highly expressed (p < 0.05); compared with ALL PreB, M(1) had high expression of HLA-DR, CD34, meanwhile myeloid antigen CD13, CD15, CD33, CD117, MPO and T-lineage antigen CD4, CD5 were all highly expressed (p < 0.05); as compared with T-ALL, the early-phase antigen HLA-DR, CD34, myeloid antigen CD13, CD15, CD33, CD117, MPO of M(1) were all significantly highly expressed (p < 0.05). In M(1), the complete remission (CR) rate in patients with CD7 positive had no statistical difference from that in patients with CD7 negative (p > 0.05); the CR rate of patients with CD34 positive had no statistical difference from that of patients with CD34 negative (p > 0.05); CR rate in M(1) was lower than that in M(2) (p < 0.05), time to reach CR was longer, the incidence of hyperleukocytic acute leukemia was higher (p < 0.05), CR rate in hyperleukocytic acute leukemia was lower (p < 0.05). It is concluded that the myeloid antigen CD33, CD13 in M(1) are highly expressed, early-phase antigen HLA-DR in M(1) is also highly expressed, but the myeloid antigen CD11b, CD15, MPO, CD117 in M(1) are lowly expressed, T-lineage antigen CD4, CD7 in M(1) are highly expressed in the meantime. There is no definite characteristic marker in immunology to differentiate M(1) from M(2), but as the positive rate of CD11b, CD15, MPO, CD117 in M(1) are significantly lower than that of M(2), CD11b, CD15, MPO, CD117 can be used as reference indicators to differentiate M(1) from M(2). AML M(1), ALL ProB, ALL PreB and T-ALL, which are difficult to differentiate in morphology can be well seperated through the analysis of immunological phenotype. CD117 is mainly expressed in AML, which is useful for the differentiation diagnosis between AML and ALL. The prognosis of M(1) is worse than that of M(2).
Adolescent ; Adult ; Antigens, CD ; analysis ; Antigens, CD7 ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; CD13 Antigens ; analysis ; CD4 Antigens ; analysis ; Diagnosis, Differential ; Female ; HLA-DR Antigens ; analysis ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; classification ; diagnosis ; immunology ; Male ; Middle Aged ; Prognosis ; Sialic Acid Binding Ig-like Lectin 3 ; Young Adult

In order to investigate the potential of human ERMAP gene in erythroid cell differentiation, K562 cells were induced to erythroid lineage by Ara-C and to macrophage lineage by TPA, human ERMAP mRNA was detected by fluorescent quantitative PCR. The results showed that human ERMAP mRNA increased while K562 cells were induced to erythroid lineage after treatment with Ara-C at 2.5 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. Human ERMAP mRNA not changed while K562 cells were induced to macrophage lineage after treatment with TPA at 2.0 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. It is concluded that human ERMAP gene plays an important role in differentiation and proliferation of erythroid cells.
Antigens, CD ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Blood Group Antigens ; genetics ; Butyrophilins ; Cell Differentiation ; drug effects ; genetics ; Cytarabine ; pharmacology ; Erythrocytes ; cytology ; metabolism ; ultrastructure ; Flow Cytometry ; Gene Expression ; drug effects ; Humans ; K562 Cells ; Macrophages ; cytology ; metabolism ; ultrastructure ; Microscopy, Electron ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Transferrin ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sialic Acid Binding Ig-like Lectin 3 ; Tetradecanoylphorbol Acetate ; pharmacology ; Time Factors

To evaluate the significance of immunologic classification for typing of acute leukemia (AL). 68 cases of AL were classified by morphologic and immunologic typings. The results showed that the consistency rate was 94.1% between morphology and immunology, and 4 morphologic misdiagnosed cases were corrected by immunology; CD13 and CD33 were special myeloid lineage-associated antigens; AML-M(3) was often CD34 low-expressed and HLA-DR-negative; CD14 was often expressed in AML-M(4) and M(5); lymphoid lineage-associated antigens (CD7) were easily found in ANLL, and myeloid lineage-associated antigens were also found in ALL. In conclusion, immunologic classification can improve the accuracy in acute leukemia diagnosis. The diagnosis of some special AL, such as acute unidentified leukemia (AUL), AML-M(0) and so on, must rely on immunologic classification.
Adolescent ; Adult ; Aged ; Antigens, CD ; biosynthesis ; Antigens, CD34 ; biosynthesis ; Antigens, CD7 ; biosynthesis ; Antigens, Differentiation, Myelomonocytic ; biosynthesis ; CD13 Antigens ; biosynthesis ; Female ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; classification ; immunology ; Lipopolysaccharide Receptors ; biosynthesis ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; classification ; immunology ; Sialic Acid Binding Ig-like Lectin 3

OBJECTIVETo investigate the application of flow cytometry in the differential diagnosis of lymphoma/leukemia with aberrant antigen expression.

METHODSThe results of flow cytometry of 30 lymphoma/leukemia cases with aberrant antigen expression, of which 3 cases being lymphomas, 8 B-cell leukemia, 1 T-cell leukemia, 17 acute non-lymphoid leukemia and 1 acute non-lymphoid leukemia involving lymph nodes were analyzed. Immunohistochemistry (EnVision) for CD79a, CD3 and MPO was performed on all cases.

RESULTSEleven cases of B-cell lymphoma/leukemia were cytoplasmic CD79a (cCD79a)-positive, cytoplasmic CD3 (cCD3epsilon) and cytoplasmic MPO (cMPO)-negative. Five of these cases were positive for CD5 and 2 for CD5, 1 or 2 for myeloid marker(s). The T-cell leukemia cases were cCD3epsilon-positive, cCD79a and cMPO-negative, they also co-expressed CD13 and CD33. The mantle cell lymphoma cases were positive for CD3, CD13 and CD33. Of the 8 B-cell leukemia cases, 4 were positive for CD5, 3 for CD13 and 1 for CD13 and CD33. The 18 acute non-lymphoid leukemia cases (including 1 acute non-lymphoid leukemia case involving lymph nodes) were cMPO-positive and cCD79a and cCD3epsilon-negative. Eight of the 18 expressed T-cell markers (including 1 case of acute non-lymphoid leukemia involving lymph nodes), 8 expressed B-cell markers, 2 expressed both T and B-cell markers.

CONCLUSIONSFlow cytometry can demonstrate aberrant antigen expression in lymphoma/leukemia cells and is helpful in delineating their cell origin. The technique is thus useful in the differential diagnosis of lymphoma/leukemia.

Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; CD13 Antigens ; metabolism ; CD3 Complex ; metabolism ; CD5 Antigens ; metabolism ; CD79 Antigens ; metabolism ; Diagnosis, Differential ; Flow Cytometry ; Humans ; Leukemia, B-Cell ; diagnosis ; immunology ; Leukemia, T-Cell ; diagnosis ; immunology ; Lymphoma, Mantle-Cell ; diagnosis ; immunology ; Peroxidase ; metabolism ; Retrospective Studies ; Sialic Acid Binding Ig-like Lectin 3