[Establishment of 14-Color Panel for Determining Leukocyte Subsets in Human Peripheral Blood with Flow Cytometry].

OBJECTIVE: To establish a 14-color flow cytometry protocol for the examination of leukocyte subsets in human peripheral blood. METHODS: We used cell membrane surface antibodies CD45, CD3, CD4, CD8, CD19, CD56, CD16, CD14, CD25, CD127, HLA-DR, CD123, CD11c and nucleus staining dye DAPI to establish a 14-color flow cytometry assay to determine the major cell subsets in human peripheral blood. We collected peripheral blood specimens from healthy volunteers to test for antibody titers and optimal photomultiplier tube (PMT) voltage, and to conduct single-color staining and fluorescence minus one control staining. After determining the test method and test conditions, the peripheral blood samples of 18 healthy volunteers were analyzed. RESULTS: According to the cell classification and staining index, optimal antibody mass concentrations selected were as follows: CD25 and CD127 at 8.0 µg/mL, CD45, CD3, CD14 and CD123 at 4.0 µg/mL, CD8, CD19, CD56, CD16, HLA-DR and CD11c at 2.0 µg/mL, CD4 at 1.0 µg/mL and DAPI at 0.1 µg/mL. The detection voltages for CD45, CD3, CD4, CD8, CD19, CD56, CD16, CD14, CD25, CD127, HLA-DR, CD123, CD11c and DAPI were 450 V, 410 V, 400 V, 550 V, 405 V, 500 V, 520 V, 550 V, 550 V, 400 V, 450 V, 400 V, 580 V, and 300 V, respectively. The appropriate fluorescence compensation was determined by single-color staining and fluorescence minus one controls. The 14-color flow cytometry panel was established to analyze the main subsets of leukocytes in human peripheral blood, and peripheral blood samples from 18 healthy adults were examined, obtaining the percentages of each subset of peripheral blood leukocytes and the immunophenotypes of the main subsets. CONCLUSION: We established a 14-color panel for determining leukocyte subsets in human peripheral blood by flow cytometry, which produced stable and reliable results and was easy to operate.

MicroRNA-184 Modulates Human Central Nervous System Lymphoma Cells Growth and Invasion by Targeting iASPP.

Central nervous system lymphoma (CNSL) remains a diagnostical and therapeutical challenge. MiRNAs post-transcriptionally regulate expression of targeted mRNAs through binding to their 3' UTR to inhibit their translation or promote their degradation. Oncoprotein inhibitory member of the ASPP family (iASPP), a key inhibitor of tumor suppressor p53, has been reported to play oncogenic role in cancers. Our present study was aimed to determine whether the miR-184/iASPP axis is involved in the proliferation and invasion of CNSL. A reduced level of miR-184 was observed in CNSL tissues. Exogenous miR-184 inhibited cell survival and invasion, as well as the tumor volumes, while miR-184 inhibition could reverse this process. The RNA and protein levels of iASPP were significantly inhibited by miR-184, and the 3' UTR of iASPP was shown to be a target of miR-184. The expression of iASPP was up-regulated in CNSL tissues, compared to that of the normal brain tissues. The inhibition of iASPP by shRNA iASPP significantly repressed CNSL cells' proliferation and invasion, and reduced the volume of the tumor. Besides, iASPP overexpression could partly restore the suppressive effect of miR-184 on CNSL cell proliferation and invasive capability. We also revealed that miR-184/iASPP axis regulated the proliferation and invasion via PI3K/Akt signaling pathway, which presents a novel potential therapy for intervention of CNSL. Taken together, our findings revealed the detailed role of the miR-184/iASPP axis in CNSL and this axis might modulate the proliferation and invasion of CNSL via regulating the PI3K/Akt signaling pathway. J. Cell. Biochem. 118: 2645-2653, 2017. © 2017 Wiley Periodicals, Inc.

[Using 9-color Flow Cytometry Immunophenotyping to Detect Activation and Apoptosis of Human TCR Vß Lymphocyte Subpopulations in Peripheral Blood Samples].

OBJECTIVE: To establish an assay using 9-color flow cytometry immunophenotyping to detect activation and apoptosis of human TCR Vß lymphocyte subpopulations in peripheral blood samples. METHODS: We used 5 antibodies (CD3, CD4, CD8, CD95, CD69), phospholipids binding proteins Annexin V, TCR Vß Repertoire Kit and nucleus dye DAPI to establish a 9-color flow cytometry assay. Peripheral blood samples were taken from eight healthy people for test of antibodies and determination of optimal PMT and staining method (single-stained vs stained with all but one antibody). RESULTS: Appropriate detecting voltage, antibody concentration and compensation methods were determined. The distribution of TCR Vß subgroup in our samples was consistent with the TCR Vß Repertoire Kit instruction and other published literature. CONCLUSION: We have established a effective easy using 9-color flow cytometry immunophenotyping to detect human TCR Vß lymphocyte subpopulations in peripheral blood samples.

Individualized leukemia cell-population profiles in common B-cell acute lymphoblastic leukemia patients.

Immunophenotype is critical for diagnosing common B-cell acute lymphoblastic leukemia (common ALL) and detecting minimal residual disease. We developed a protocol to explore the immunophenotypic profiles of common ALL based on the expression levels of the antigens associated with B lymphoid development, including IL-7Rα (CD127), cytoplasmic CD79a (cCD79a), CD19, VpreB (CD179a), and sIgM, which are successive and essential for progression of B cells along their developmental pathway. Analysis of the immunophenotypes of 48 common ALL cases showed that the immunophenotypic patterns were highly heterogeneous, with the leukemic cell population differing from case to case. Through the comprehensive analysis of immunophenotypic patterns, the profiles of patient-specific composite leukemia cell populations could provide detailed information helpful for the diagnosis, therapeutic monitoring, and individualized therapies for common ALL.

[Effect of cyclooxygenase-2 inhibitor on CD4+ CD25+ regulatory T cells in mouse hepatocellular carcinoma].

OBJECTIVE: To investigate the effect of celecoxib on regulatory T cells (Treg) in mouse hepatocellular carcinoma (HCC). METHODS: Total of 40 mice was divided into two subgroups, normal animal groups include control and celecoxib group, HCC groups include control and celecoxib group. 30 mg/kg of celecoxib were given daily for 24 days for celecoxib groups. All mice were sacrificed after 24 days treatment and the removed tumor weight were measured. By detecting CD4 and CD25 with flow cytometry, the level of Treg in peripheral blood was determined. The expressions of Forkhead/winged helix transcription factor-3 (Foxp3) protein in the tumor infiltrating lymphocytes (TILs) and cyclooxygenase-2 (COX-2) protein in tumor tissue were measured by immunohistochemistry techniques. RESULTS: The mean weight of tumor in celecoxib group is much lower than that of control group [(0.82 +/- 0.30) g vs. (1.41 +/- 0.63) g, P < 0.05]. The percentage of Treg in total CD4+ T cells isolated from the peripheral blood of HCC animals in control group was higher than that of normal control group [(4.26 +/- 0.89)% vs. (3.01 +/- 0.65)%, P < 0.05]. After treatment with celecoxib, the percentage of Treg was decreased [(3.04 +/- 0.74)% vs. (4.26 +/- 0.89)%, P < 0.053 and the percentage of Foxp3 positive cell in TILs was also decreased [(8.87 +/- 3.72)% vs. (30.78 +/- 9.26)%, P < or = 0.05]. The tumor tissue COX-2 protein expression in celecoxib group was lower than in that of control group [IOD (2.90 +/- 1.030) vs. (6.63 +/- 2.279), P < 0.01)] and the changing of COX-2 in tumor tissue was according to Treg in the peripheral blood. CONCLUSION: Treg cells are increased in the peripheral blood of HCC mice and COX-2 inhibitor could decrease the percentage of Treg cell in the peripheral blood or TILs.

Growth-inhibitory and apoptosis-inducing effects of tanshinones on hematological malignancy cells and their structure-activity relationship.

This study has investigated the growth-inhibitory and apoptosis-inducing effects of dihydrotanshinone, tanshinone I, tanshinone IIA, and cryptotanshinone on hematological malignancy cell lines, aiming to explore their structure-activity relationship. The growth-inhibitory effects of the tanshinones on K562 and Raji cells were assessed using a modified MTT assay; the apoptosis-inducing effects were assessed by fluorescence microscopy and flow cytometry analysis. The changes in cellular morphology were observed using an inverted phase-contrast microscope. MTT results revealed that these tanshinones inhibited cell proliferation in a concentration-dependent and time-dependent manner. The IC50 values of dihydrotanshinone, tanshinone I, tanshinone IIA, and cryptotanshinone for K562 cells were 3.50, 13.52, 19.32, and 47.52 µmol/l at 24 h; 1.36, 4.70, 5.67, and 22.72 µmol/l at 48 h; and 1.15, 1.59, 2.82, and 19.53 µmol/l at 72 h, and the values for Raji cells were 3.30, 4.37, 12.92, and 52.36 µmol/l at 24 h; 1.55, 1.71, 6.54, and 25.45 µmol/l at 48 h; and 1.07, 1.38, 1.89, and 18.47 µmol/l at 72 h. The flow cytometry analysis demonstrated that these tanshinones induced apoptosis of K562 cells in a concentration-dependent manner, and dihydrotanshinone as well as tanshinone I were more potent than tanshinone IIA and cryptotanshinone. Some noticeable apoptotic morphologies could be observed by fluorescence microscopy on tanshinones-treated Raji cells. Collectively, these tanshinones caused growth inhibition and apoptosis in hematological malignancy cell lines, with dihydrotanshinone being the most potent, followed by tanshinone I, tanshinone IIA, and cryptotanshinone. These results suggested that the structure of aromatic ring A enhanced the cytotoxicity and the structure of ring C may have contributed to the cytotoxicity, but the mechanisms need to be further investigated.

[Effect of diagnostic ultrasound-mediated microbubble contrast and urokinase on augmentation thrombolysis and optimization of the major parameters: an in vitro study].

OBJECTIVE: To investigate the efficacy of diagnostic ultrasound and microbubble contrast (MB) on enhancing thrombolysis in combination with urokinase (UK) and to determine the optimal combination for thrombolysis in vitro. METHODS: Four types of standardized red thrombus were prepared in vitro, including 3-hour-old (3 h), 6-hour-old (6 h), 12-hour-old (12 h), and 24-hour-old (24 h). The major parameters for the designed experiments included transmit powers of ultrasound (factor A, 5%, 25%, 50%, 100%), MB volumes (factor B, 50 microL, 100 microL, 200 microL, 400 microL), UK concentrations (factor C, 100 U/mL, 200 U/mL, 400 U/mL, 800 U/mL), and lysis time (factor D, 10 min, 20 min, 30 min, 40 min). An orthogonal array experimental design (OAD) based on four levels L16 (4(5)) of the above four parameters was employed to optimize the thrombolysis conditions. During the procedure of thrombolysis, the diagnostic ultrasound frequency was fixed at 1.82 MHz. The histopathological changes measured by HE staining and scanning electron microscope (SEM) were carried out to observe the clots before and after thrombolysis. The loss of clot weight before and after treatment was measured to determine the lysis efficiency (LE). Analysis of variance (ANOVA) was performed to assess the LE according to the L16 (4(5)) matrix. RESULTS: The HE staining and SEM observation of thrombolysis under the following experimental conditions of 5% ultrasound transmit power, 400 microL MB volume, 800 U/mL UK concentration, and 40 min lysis time showed remarkable disaggregation of fibrin nets. The above four factors had significant impact on thrombus (all P < 0.05), among which UK concentrations (factor C) was the most significant one. The optimal scheme was determined as a C4-D4-A1-B4 mode, with UK concentration 800 U/mL, lysis time 40 min, transmit power 5%, and MB volume 400 microL, respectively. The LE curves for 3h clots were superior to the others. The lysis efficiencies for the clots showed significant differences among different type of thrombus (all P < 0.05). CONCLUSION: 1.82 MHz diagnostic ultrasound and microbubble contrast can be applied to augment thrombolysis in vitro even with a transmit power as low as 5%. Under the condition of fixed ultrasound frequency, the LE of thrombus increase with increased UK concentrations, lysis time and MB volumes, and decrease with increased thrombus ages.

[The value of von Willebrand factor pro-peptide and ADAMTS13 in diagnosis of thrombotic thrombocytopenic purpura].

OBJECTIVE: To evaluate the combined analysis of ADAMTS13 activity and von Willebrand factor (vWF) pro-peptide level in the diagnosis and treatment of thrombotic thrombocytopenic purpura (TTP). METHODS: ADAMTS13 activity was measured by Fluorescenced substrate method (with Frets-vWF73), and vWF pro-peptide was measured by sandwich ELISA in 11 patients with idiopathic TTP, 5 patients with secondary TTP, 2 patients with transplantation associated TTP and 3 patients with suspected TTP. Plot each patient's data in the graph with coordinates of ADAMTS13/vWF pro-peptide. The plot was divided into 4 quadrants by 2 lines, one went through 10% in ADMATS13 axis, and the other went through 3 in vWF pro-peptide axis. Analyze the characteristics of points in different quadrants. RESULTS: Mean ADAMTS13 activities of idiopathic, secondary, transplantation associated and suspected TTP were 6.90%, 3.88%, 13.2% and 19.46% respectively. Mean times of elevated vWF pro-peptide from normal plasma pool of idiopathic, secondary, transplantation associated and suspected TTP were 4.2, 3.2, 4.5 and 2.9 respectively. In ADAMTS13/vWF pro-peptide figure, 57.1% of patients with TTP were in quadrant III, IV, all patients with transplantation associated TTP were in quadrant II ,2 of 3 patients with suspected TTP were in quadrant I. CONCLUSION: Combined analysis of ADAMTS13 and vWF pro-peptide may provide clues and advices in the diagnosis and treatment of TTP, especially for the suspected ones.

[Antitumor effects of matrix protein of vesicular stomatic virus on EL4 lymphoma mice].

OBJECTIVE: To explore antitumor effects of plasmid pcDNA3. 1-MP encoding matrix protein of vesicular stomatitis virus (VSV) complexed with cationic liposome (DOTAP:CHOL) in mice with EL4 lymphoma. METHODS: C57BL/6 mouse model with EL4 lymphoma was established. Sixty mice bearing EL4 lymphoma were divided randomly into five groups including Lip-MP, Lip-pVAX, Lip, ADM and NS groups, which were intravenously injected with liposome-pcDNA 3. 1-MP complex, liposome-pVAX complex, empty liposome, Adriamycin and normal saline respectively every three days. Tumor volumes and survival time were monitored. Microvessel density and tumor proliferative index in tumor tissues were determined by CD31, Ki-67 immunohistochemistry staining, meanwhile the tumor apoptosis index was measured by TUNEL method. RESULTS: From 6 days after treatments on, the tumor volume in Lip-MP group was much smaller than that in Lip-pVAX, Lip and NS group (P < 0.05). The median survival time of mice in Lip-MP group, 44 days after inoculation of tumor cells, was significantly higher than that in other groups (P < 0.05), which was 39 days, 38.5 days and 34 days in Lip-pVAX, Lip and NS groups respectively. The MVD value in tumor tissues in Lip-MP group was less than that in Lip-pVAX, Lip and NS groups (P < 0.05). Ki67 staining revealed that Lip-MP complex apparently suppressed the proliferation of EL4 tumor cells in vivo (P < 0.05). TUNEL assays showed that apoptosis index of tumor cells in Lip-MP group, 10.60 +/- 1.71, was much higher than that in other three groups (P < 0.05). CONCLUSIONS: Lip-MP complex, the plasmid encoding matrix protein of VSV (VSV-MP) encapsulated in cationic liposome, significantly inhibited the growth of tumor and prolonged the survival of mice bearing EL4 lymphoma, which may be related to the induction of tumor cell apoptosis, inhibition of tumor angiogenesis, and suppression of tumor cell proliferation.

Association of the interferon-gamma single nucleotide polymorphism +874(T/A) with response to immunosuppressive therapy in patients with severe aplastic anemia.

The aim of this study was to investigate whether a single nucleotide polymorphism (SNP) +874T/A of interferon-gamma (IFN-γ) correlates with response to immunosuppressive therapy. Amplification refractory mutation system-polymerase chain reaction was used to amplify the polymorphic segments of the IFN-γ +874T/A gene in the samples obtained from 54 patients with aplastic anemia and 51 healthy adults. Further, enzyme linked immunosorbent assay was used to assay IFN-γ levels in the blood plasma of 35 patients with severe aplastic anemia before immune suppression therapy and 20 healthy blood donors. The results showed that the frequency of IFN-γ +874 TT genotype in patients with aplastic anemia was significantly higher than the corresponding frequency in the healthy adults (42.6% vs. 17.6%, χ(2)=13.78, p=0. 01). The response rate in severe aplastic anemia patients with increased IFN-γ levels in the blood plasma was higher than that in severe aplastic anemia patients with decreased IFN-γ levels in the blood plasma (73.7% vs. 25.0%, p<0.05). Of the 35 patients with severe aplastic anemia, 15 showed the IFN-γ +874 TT genotype, whereas response in 11 patients, the high response rate was significantly in the favor of the IFN-γ +874 TT genotype (73.3% vs. IFN-γ +874 non-TT 35%, p<0.05). In conclusion, the IFN-γ +874T/A gene polymorphism may be correlated with response to immunosuppressive therapy.

[Apoptosis of NB4 cells induced by Tanshinone II A combined with arsenic trioxide].

OBJECTIVE: To evaluate the synergistic effects of Tanshinone II A (Tan II A) combined with arsenic trioxide (ATO) on apoptosis and differentiation of NB4 cells. METHODS: The NB4 cells were treated with Tan II A, ATO, and Tan II A combined with ATO, respectively. The proliferative inhibition rates of NB4 cells induce by Tan II A, ATO and their combination were calculated on the fifth day. The morphology of cell differentiation was observed by Giemsa stain and transmission electron microscope (TEM). The cell cycle, proliferation index and apoptosis induced by these drugs were measured by flow cytometry (FCM). RESULTS: 1) Combination treatment of Tan II A with ATO had synergistic effects on the proliferative inhibition of NB4 cells. This synergistic effects showed a dose and time-dependent manner. The proliferative inhibition rates of NB4 cells were increased gradually after the treatment of 1.0 microg/mL Tan II A combination with 0.125 micromol/L, 0.25 micromol/L and 0.5 micromol/L ATO respectively, which was much higher than those treated by corresponding ATO alone (P < 0.01); 2) Combination treatment of Tan lI A with ATO had synergistic effects on the apoptosis of NB4 cells. The synergistic effects of 1.0 microg/mL Tan II A combined with 0.5 micromol/L ATO on the apoptosis of NB4 cells lasted for 3 days; 3) Combination treatment of Tan II A with ATO had synergistic inhibition effects on proliferation of NB4 cells, which decreased the number of cells in S phase and increased cells in G0/G1 phase. The proliferation indices (PI) of NB4 cells treated with the combination of Tan II A and different level of ATO were lower than those treated by corresponding ATO alone (P < 0.05). Combination treatment of Tan II A with ATO had no significant synergistic effects on the differentiation of NB4 cells. CONCLUSION: Combination treatment of Tan II A with ATO has significant synergistic effects on the apoptosis of NB4 cells. This synergistic effects is related to the dosage and treatment time. The significant synergistic effects of Tan II A and ATO on NB4 cells differentiation is not demonstrated in our experiment.

[Effects and mechanisms of recombinant human erythropoietin in treating multiple myeloma in murine mice].

OBJECTIVE: To test the effects and to identify appropriate dosage and possible mechanisms of recombinant human erythropoietin (rHuEPO) in treating MPC-11 myeloma in the BALB/c murine models. METHODS: A total of 420 BALB/c mice were divided into five groups. 410 of which were injected with 10(4) MPC-11 cells. The 10 mice without myeloma cell inoculation served as normal controls. On the fifth day after inoculation, 50 tumor-bearing mice were arbitrarily assigned into the placebo group, while the other 360 mice were randomly allocated into three experimental groups. Each experimental group had 120 mice and received 10, 20 and 30 units subcutaneous injection of rHuEPO, respectively. A daily injection was administered for 14 days, followed by three injections per week. The mice in the placebo group were administered with saline following the same scheme. Dynamic monitoring of serum M-protein and hemoglobin levels of the mice were performed after myeloma cell inoculation. The subcutaneous nodules were sent for pathological biopsy to ascertain the successful establishment of the murine models. The mice were randomly chosen from each group to be tested for the levels of monoclonal IgG and kappa light chain in their sera (immunofixation electrophoresis and ELISA), counts of CD4 and CD8 positive cells in whole blood (flow cytometry), microvessel density (by marking CD31) and cell apoptosis (TdT-mediated dUTP-biotin nick end labeling, TUNEL) of tumor tissues, as well as the levels of cytokines such as IL-6 and TNF-alpha in sera (ELISA) at each month after the injection of rHuEPO. RESULTS: The serum monoclonal immunoglobulin appeared on the 22nd day after inoculation of MPC-11 cells. rHuEPO increased Hb level and survival time of the mice with multiple myeloma. The serum levels of IL-6, IgG and kappa light chain decreased significantly in the mice in the treatment groups compared with those in the placebo group. The overall survival time showed a positive correlation with the Hb level (P = 0.000), and a negative correlation with the serum levels of IL-6 (P = 0.009). CONCLUSION: rHuEPO increases Hb levels and survival time and reduces serum IL-6 and M-protein of the mice with multiple myeloma.

[Gene cloning of mIFN-gamma and construction of recombinant adenovirus].

OBJECTIVE: To develop a rapid and efficient method for preparing recombinant adenovirus containing mouse IFN-gamma (mIFN-gamma) gene by homologous recombination in E. coli. in order to build a foundation for research into gene therapy of liver fibrosis. METHODS: The target gene mIFN-gamma was amplified by using PCR from the vector pORF5-mIFN-gamma. Once verified, it was cut out by double endonucleases, then connected to the shuttle vector pAdTrack-CMV. The newly constructed vector was linearized by Pme I following transformation to the E. coli. BJ5183, which contained the backbone vector pAdEasy-1. The correct recombinant pAd-mIFN-gamma was selected by endonucleases and by Kanamycin resistance. Again it was linearized with Pac I , then transfected to AD-293 cells by means of Calcium Phosphate method. Finally, the target gene IFN-gamma was identified by PCR and Western blot methods. RESULTS: The target gene mIFN-gamma amplified by PCR was identified by DNA sequencing, which proved that the mIFN-gamma gene consisted of 468 nucleotides and was completely the same with the sequence published on the GenBank. The adenoviral vector constructed by homologous recombination had the gene of interest and the viral could be examined 4-6 days after transfection, and the green fluorescence intensity became greater at about 8-11 days. The adenovirus obtained at the 12th day was digested by protease K and then was amplified by PCR and identified by Western blot. The two methods proved that the adenovirus encoded the target gene mIFN-gamma. CONCLUSION: Preparing recombinant adenovirus containing mIFN-gamma gene by homologous recombination in E. coli. Is a rapid and efficient method. The Ad-mIFN-gamma can be propagated in 293 cell line. It may be used as a novel agent for gene therapy in liver fibrosis.

[Effects of urokinase intervention on endotoxin-induced DIC in Wistar rat model].

OBJECTIVE: To study the effects of urokinase intervention on endotoxin-induced DIC in Wistar rats model. METHODS: Wistar rats were randomly assigned into 4 groups: normal saline (NS) group, urokinase (UK) group, endotoxin (LPS) group and LPS+ UK group. These agents were given to the rats by the tail vein intravenous infusion, NS group was treated with NS 2.5 mL/h x 4 h; UK group with NS 2.5 mL/h, 1 h later UK 4 IU/(g x h) x 3 h; LPS group with LPS 3 mg/(kg x h) x 4 h; LPS+UK group with LPS 3 mg/(kg x h) firstly, 1 h later UK 4 IU/(g x h) ) < 3 h. After the intervention, the function of coagulation and fibrinolysis, the indicators of organ damage and microcirculation fibrin micro-clots were evaluated. RESULTS: One hour after the infusion of 3 mg/(kg x h) of LPS, DIC signs began to appear, and became more apparent over time. After the intervention of urokinase, the values of clotting time (PT), activated partial thromboplastin time (APTT) were significantly shorter, but the platelet count (PLT), the amount of fibrinogen (FIB) changed little. Plasminogen activator inhibitor-1 (PAI-1) level decreased, while the D-dimer level increased. Serum creatinine (Cr), alanine aminotransferase (ALT) also decreased significantly. The biopsy of liver, kidney, and lung showed tissue damage became better, and the organ microcirculation fibrin micro-clots decreased significantly. CONCLUSION: The concentration of 3 mg/(kg x h) endotoxin can successfully induce DIC model in Wistar rats. Urokinase could play a positive role to prevent the LPS-induced DIC.

[Effect of tanshinone II A on NB4 cell induced procoagulant activity in human umbilical vein endothelial cells].

OBJECTIVE: To investigate the effect of tanshinone II A on the procoagulant activity (PCA) of human umbilical vein endothelial cells (HUVEC) induced by acute promyelocytic leukemia (APL) cell line NB4 cells. METHODS: The HUVEC were incubated for 6, 12, and 24 hours in different tanshinone II A conditioned medias (Tan II A-NB4-24h-CM, Tan II A-NB4-72h-CM, Tan II A-NB4-120h-CM). Then the HUVEC were incubated for 6, 12, 24, and 72 hours with Tan II A-NB4-120h-CM and different concentrations of Tan II A (0, 0.25, 0.5, 1.0 microg/mL). The HUVEC lysates were obtained by three repeated freezing and thrawing. Their PCA were tested using the one stage clotting assay. The activity of tissue factor (TF : act) was tested using the chromogenic substrate assay. The control groups included 0.3 microg/mL ATRA, 0.01% DMSO and RPMI 1640. RESULTS: Tan II A-(72 h,120 h)-NB4-CM elevated PCA of HUVEC and six hours of incubation in the 120 h-NB4-CM had the greatest PCA. The PCA of HUVEC in the 1.0 microg/mL Tan II A-NB4-CM was the same as in the 0.3 microg/mL ATRA-NB4-CM. (2) The NB4-CM induced PCA of HUVEC decreased with 5.0 microg/mL of Tan II A, at a level similar to the decrease with 0.3 microg/mL of ATRA. Less than 5.0 microg/mL of Tan II did not reduce the NB4-CM induced PCA of HUVEC. (3) Both Tan II A 120 h-NB4-CM and ATRA 120 h-NB4-CM elevated the TF : act of HUVEC. The TF : act reached the peak after 6 hours of incubation. The Tan II A 120 h-NB4-CM maintained the peak level of TF : act at the 12th hour and fell to the base line at the 24th hour. The ATRA 120 h-NB4-CM induced TF:act dropped down with time after reaching its peak at the 6th hour. (4) The 1.0 microg/mL of Tan II A did not reduce the TF : act of HUVEC induced by the Tan II A 120 h-NB4-CM. But the 0.3 microg/mL of ATRA reduced the TF : act of HUVEC at the 6th hour. CONCLUSION: TanIIA-NB4-CM increases PCA and TF : Act of HUVEC. TanIIA decreases PCA of HUVECs induces by TanIIA-NB4-CM.

[Inhibitive effect of artesunate on human lymphoblastic leukemia/lymphoma cells].

OBJECTIVE: To test the effect of Artesunate (ART) on the proliferation of Raji cells, Jurkat cells and acute lymphoblastic leukemia (ALL) primary cells; to determine the synergistic antiproliferation effect between ART and Vincristine (VCR) or Cytarabine(Ara-C) on Raji and Jurkat cells; and to explore the mechanism of ART induced apoptosis of tumor cells in vitro. METHODS: MTT assay was performed to detect the inhibition of proliferation of Raji, Jurkat, and ALL primary cells. The cells were exposed to ART at various concentrations with or without VCR or Ara-C. The morphological changes of Raji and Jurkat cells were observed under light microscopy after Wright-Giemsa dyeing and electron transmission microscopy. The mitochondria transmenbrane potential was measured by Rhodamine 123 staining. Colorimetric method was used to measure the activities of caspase-3 in those tumor cells. RESULTS: ART inhibited the proliferation of Raji cells, Jurkat cells and ALL primary cells. The cytotoxicity of ART on Raji cells and Jurkat cells at a low concentration increased when combined with VCR or Ara-C. Apoptosis in Raji cells and Jurkat cells appeared after exposure to ART. Raji cells and Jurkat cells exposed to ART showed mitochondria transmembrane potential collapse. ART increased the caspase-3 activities of Raji, Jurkat and ALL primary cells. CONCLUSION: ART alone or combined with chemotherapy drugs could inhibit the proliferation of B/T lymphocytic tumor cell lines as well ALL primary cells in vitro, probably through the mechanism of apoptosis, which suggest that ART is likely to be a potential drug in the treatment of leukemia/lymphoma.

[Expression of hOCT1 in patients with chronic myelogeneous leukemia treated by imatinib mesylate].

OBJECTIVE: To measure the expression of hOCT1 gene in patients with Chronic Myelogeneous Leukemia (CML) and to explore its role in the progress of the disease and responding to Imatinib Mesylate (IM) treatment. METHODS: Sixty three peripheral blood or bone marrow samples were taken from 30 patients with CML (20 in chronic phase, 10 in advanced phase). The samples were divided into two groups: responding (optimal and suboptimal) and non-responding according to effectiveness of the IM treatment. The mRNA levels of hOCT1 gene were detected with RT-PCR (SQ-PCR), 3, 6, 9, 12 and 15 months after the IM therapy. The associations between hOCT1 gene levels and clinical presentations, laboratory indicators and cytogenetic findings were analysed. RESULTS: No significant difference in the expression levels of hOCT1 gene was found before and after the IM treatment (0.5110+/-0.1629 vs 0.5207+/-0.1909, P=0.5840). No significant difference in the expression levels of hOCT1 genes was found between the patients in chronic phase and the patients in advanced phase before the IM treatment (0.5525+/-0.1985 vs 0.4490+/-0.1717, P=0.1090). The levels of hOCT1 did not have significant changes 3, 6, 9, 12 and 15 months after the IM treatment (P=0.3412). No significant difference in the expression levels of hOCT1 genes was found between the 15 patients with optimal and suboptimal responding to IM and the 5 patients with no responding to IM (0.5820+/-0.1460 vs 0.4640+/-0.1781, P=0.127). Although the hOCT1 levels of the 16 chronic patients increased after IM treatment compared to the baseline (0.5207+/-0.1909 vs 0.5110+/-0.1629, P=0.001), there was no significant correlation between the increase of hOCT1 and the decrease of BCR-ABL (P=0.821). CONCLUSION: hOCT1 has no association with the stage and course of CML, nor with the effectiveness of IM therapy.

[Studies on the predictors of response to immunosuppressive therapy in severe aplastic anemia patients].

OBJECTIVE: To assay the factors that may predict the response to immunosuppressive therapy (IST) in severe aplastic anemia patients. METHODS: The blood samples were collected from 37 patients diagnosed as severe aplastic anemia in West China Hospital of Sichuan University and West China Second Hospital of Sichuan University during February, 2006 to March, 2007. Twenty healthy blood donors were used as normal control. The plasma levels of IFN-gamma and IL-2 were measured by enzyme linked immunosorbent assay, and the gene phenotype of HLA-DRB1 * 15 and HLA-DRB1 * 1501 was analyzed by polymerase chain reaction with sequence specific primer. The expressions of CD55 and CD59 on the cellular membrane of red blood cell and white blood cells also were measured. RESULTS: The response rate in the patients who had higher IL-2 level before IST was significantly better than that in the patients with lower IL-2 level (66.7% vs 28.6%, P<0.05), while similar result was observed to IFN-gamma (73.7% vs 25.0%, P<0.05). The response rate in the patients with positive HLA-DRB1 * 15 was higher than that in those negative patients (62.5% vs 44.4%, P>0.05), but there was no different found in different HLA-DRB1 * 1501 phenotypes (50% vs 50%, P>0.05). The response rates in the patients with deficient CD55 and CD59 expression were higher than those expressed CD55 and CD59 normally (80.0% vs 40.0%, P>0.05); the response rate of patients younger than 40 years was higher than those older than 40 years (60.0% vs 14.3%, P<0.05); the response rate in female patients was higher than male patients (62.5% vs 42.9%, P<0.05). CONCLUSION: The concentration of IL-2, IFN-gamma before IST, and age can be used as the predicting factors to immunosuppressive therapy, while the predicting value of HLA-DRB1 * 15, HLA- DRB1 * 1501 and CD55, CD59 to the response of IST still remain unclear.

[The polymorphism of interferon gamma gene CA short tandem repeat is associated with aplastic anemia].

OBJECTIVE: To explore the possible relationship between the polymorphism of CA short tandem repeat in first intron of interferon-gamma (IFN-gamma) gene and the susceptibility of aplastic anemia. METHODS: 54 patients, who were diagnosed as acquired aplastic anemia in West China Hospital of Sichuan University when it was from February 2006 to March 2007, and simultaneously 51 healthy adults were enrolled as normal control for this project. The polymerase chain reaction and polyacrylamide gel electrophoresis were used to assay the polymorphism of CA short tandem repeat. RESULTS: The frequency of the homozygous for 12-12 (CA) repeats or the single allele 12 (CA) repeats of the patients was 18.52% or 50.92% respectively, which was obviously higher than the control group's frequency which was 2.00% or 26.47% (P = 0.008, P < 0.001 respectively). The frequency of the homozygous for 12 (CA) repeats or the single allele 12 (CA) in acute aplastic anemia group was 12. 00% or 48.00%, while in the chronic aplastic anemia group that frequency was 24.14% or 53.45%, the statistic analysis showed there was no significant difference between those two groups (chi2 = 1.311, P = 0.252, chi2 = 0.319, P = 0.572). CONCLUSION: This study suggests that: IFN-gamma CA short tandem repeat polymorphism is associated with the susceptibility of aplastic anemia but has no relation to the severity of the aplastic anemia.

[Study of molecular mechanism of tanshinone II A inducing differentiation in acute promyelocytic leukemia NB4 cells].

OBJECTIVE: To investigate molecular mechanism of tanshinone II A inducing differentiation and apoptosis in acute promyelocytic leukemia NB4 cells. METHOD: NB4 cells were cultured in vitro and treated with tanshinone II A and observed cellular morphology, cell category and the cellular proliferation. DNA microarray technique was used to analyze the gene expression profiles of NB4 cells induced by tanshinone II A. RESULT: 92.8% of NB4 cells treated with 0.5 mg x L(-1) tanshinone II A were induced into mature neutrophils, in which myetocytes and melamyetocytes were 27.0%, banded and segmented neutrophits 68.2%. Cell growth were inhibited. cDNA microarray showed the enormously expressed 183 genes including 23 differentiation associated genes, and other interrelated genes. CONCLUSION: Tanshinone II A inducing differentiation in NB4 cells may be via regulation of many kinds of genes, especially differentiation associated genes expression. This partially explained the molecular mechanism of tanshinone II A inducing differentiation.