COX-2 inhibitor celecoxib can suppress the proliferation of FLT3-ITD positive acute myeloid leukemia cells with prominent down regulation of MEK/MCL-1 expression in vitro / 中国实验血液学杂志

The purpose of this study was to investigate the effects of Celecoxib on the proliferation of the FLT3-ITD positive and negative acute myeloid leukemia cells and its mechanism. The proliferation inhibition effect of Celecoxib with different doses on the FLT3-ITD positive cells MV4-11 and the FLT3-ITD negative K562 cells was detected by CCK-8 method, the cell apoptosis was determined by flow cytometry, and the MEK, Mcl-1, pAKT expression was tested by Western blot. The results showed that Celecoxib inhibited the proliferation of both MV4-11 and K562 cells, but the IC50 for MV4-11 was (29.14 ± 2.4) µmol/L, which was significantly lower than that of K562 cells (39.84 ± 1.0) µmol/L (P < 0.05); The induced apoptosis rate of Celecoxib at 20-80 µmol/L on MV4-11 was not observed, but there was apparent influence on K562 at the same concentration. Western blot showed that Celecoxib down-regulated the expression of MEK and Mcl-1 but did not change the expression of pAKT obviously on MV4-11 cells, while the expression of Mcl-1 was reduced a little, but no obvious change were found in the expression of MEK and pAKT on K562 cells. It is concluded that the Celecoxib can inhibit the proliferation of FLT3-ITD positive AML cells distinctly, and the potential mechanism may be related to the inhibition of the MEK/Mcl-1 signaling pathway.

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

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.

The significance of serum GM and BG antigens assay for invasive fungal infections in hematological malignancies patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the diagnostic value of serum galactomannan antigen (GM) and (1→3)-β-D-glucan antigen (BG) assay in invasive fungal infections (IFI) in the patients with hematologic malignancies and the role in monitoring therapeutic response.</p><p><b>METHODS</b>Fifty one patients with hematological malignancies met the criteria for inclusion: (1) body temperature above 38°C for 48 hours, (2) failure to respond to broad-spectrum antibiotic treatment, or (3) temperature rose again after the responded drop. Blood samples were collected twice at the first week, then once a week in at least four weeks. The double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and colorimetric assay were used for detecting GM and BG. The positive GM test is defined as two consecutive tests at different time GM value > 0.5 or > 0.8 and the positive G test is defined as BG value > 80 pg/ml. The patients were assigned into four groups as proven, probable, possible, and non-fungal infection respectively, and 21 normal volunteers were as controls.</p><p><b>RESULTS</b>Two hundred and forty serum samples were collected from 51 patients including 2 of proven IFI, 26 probable IFI, 17 possible IFI and 6 non-fungal infection. The true-positive group including the proven and probable groups, and true negative group was the non-fungal infection group. GM tests were positive in 21 of 28 cases in true positive group, and only one of 6 cases in non-fungal infection. The sensitivity, specificity, positive predictive value and negative predictive value were 75%, 83.3%, 95.5% and 41.7%, respectively. G tests were positive in all 28 cases of the true positive group, and 4 in 6 non-fungal infection cases. The sensitivity, specificity, positive predictive value and negative predictive value were 100%, 33.3%, 87.5% and 100%, respectively. G test is more sensitive than GM test (P = 0.015), but there was no significant difference in specificity of the two tests (P = 0.242). In 19 of 21 patients with GM test positive, anti-fungal treatment was effective, and GM value gradually decreased to negative, two invalid patients were persistent with GM test positive. After two weeks treatment, the average GM value was significantly lower in the effective group than in the ineffective group (P < 0.05). BG values in the responded patients showed a gradual decline similar to that of GM values, but not to negative. The changes of BG value in ineffective group varied with a trend upward. The changes in BG value had no relation with treatment effectiveness.</p><p><b>CONCLUSIONS</b>Serum GM and BG antigens detection provides strong evidence for early diagnosis of IFI. Combination of GM and G tests can improve the diagnostic specificity and reduce the false positive GM test seems superior to G test for monitoring GM and BG values during treatment.</p>

Establishment of a multiple myeloma local tumor model in mice / 中国实验血液学杂志

This study was purposed to establish a multiple myeloma local tumor model in the BLAB/c mice. Healthy BLAB/c mice were injected subcutaneously with 6 x 10(5) MPC-11 cells. In the peak time of the subcutaneous nodules observed, five mice were randomized selected to be executed and the subcutaneous nodules of these mice executed were used to detect the CD138 and kappa light chain by means of HE staining and the immunohistochemistry methods. The serum immunofixation electrophoresis (IFE) of tumor-bearing mice were performed at 5, 7, 9, 11, 12, 35 and 65 days after the initial MPC-11 cell injection. Hemoglobin level was assayed at 15 and 30 days after the initial MPC-11 cell injection. The serum levels of IL-6 were also assayed at 35 and 65 days after the initial MPC-11 cell injection. The tumor volume was monitored twice a week and their body weights were measured once a week. The results showed that the peak of the subcutaneous nodules appeared at 12 to 15 days after the initial MPC-11 cell injection. The serum monoclonal immunoglobulin could be detected at 12 days after MPC-11 cell injection. The results of HE staining and immuno-histochemistry assay for detection of CD138 and kappa light chain positive expressions proved that the subcutaneous tumor nodules originated from MPC-11 plasmacytes. The serum monoclonal protein (M protein) of the tumor-bearing mice was detected at 12 days after bearing tumor which manifested thick bands of IgG and kappa light chain. The peak time of mortality was at 20 to 40 days after the initial MPC-11 cell injection, and the median survival time was 31 days. Anemia in mice appeared at 15 days. There was a significant difference of Hb level between the tumor-bearing group and the normal group at 15 and 30 days respectively (p < 0.05). The serum level of IL-6 in tumor-bearing mice was higher than that in the normal group. It is concluded that to establish the multiple myeloma local tumor model in mice by using subcutaneous injection of MPC-11 cells has various advantages, such as simple method of model established, relative high success of bearing tumor, easy observation of tumor growth change and so on. This model can be useful for studying and evaluating the therapeutic efficacy for multiple myeloma through monitoring the changes of tumor size, serum IL-6 level and serum immunofixation electrophoresis.

Correlation of expression of PTEN with AKT phosphorylation level in leukemia cells / 中国实验血液学杂志

The study was aimed to explore the correlation of expression of pten mRNA and PTEN protein with AKT phosphorylation levels in various types of leukemia and to elucidate its role in the pathogenesis of leukemia so as to provide some evidence for using PI3K/AKT pathway inhibitors in future. 128 de novo leukemia patients were enrolled in this study, including 61 AML cases, 27 ALL cases, 24 CML cases, and 16 CLL cases. 21 volunteers were selected as normal control. The RT-PCR and Western blot were used to assay the expressions of pten mRNA, PTEN protein, and P-AKT protein in Jurkat cells, bone marrow mononuclear cells of patients respectively. The results showed that the expressions of pten mRNA and PTEN protein in Jurkat cells were lower than that in normal control group; the expression of pten mRNA in AML group was lower than that in normal control group, but the difference was not significant (p=0.274); the expressions of pten mRNA in ALL, CML, CLL each group were lower than that in normal control group, and the difference was significant (p<0.05). Compared with normal control group, the expression of PTEN protein was lower and the expression of P-AKT protein was higher in AML, ALL, CML, CLL each group, the difference were significant (p<0.05). It is concluded that the a lower expression of PTEN protein and higher expression of P-AKT protein may play an important role on leukemia pathogenesis, and inactivation of PTEN protein mainly occurs in the level of protein translation.

Expression of B7-H1 molecule on human bone marrow mesenchymal stem cells and its effects on T lymphocyte proliferation / 中国实验血液学杂志

The mechanisms of human bone marrow mesenchymal stem cells (hBMMSCs)-mediated immunomodulation are still not be completely clarified. In order to investigate the expression of B7-H1 on hBMMSCs and to explore whether B7-H1 mediated signaling pathway (B7-H1/PD-1) involves in the mechanisms of hBMMSCs-mediated immunomodulation, the hBMMSCs were isolated, cultured and identified, the B7-H1 expression on hBMMSCs was detected by flow cytometry, RT-PCR, and Western blot. The inhibitory effect of hBMMSCs on proliferation of T lymphocytes was observed in mixed lymphocyte culture, and then the functional anti-B7-H1 monoclonal antibody (mcAb) was used to block B7-H1, the proliferation of T lymphocytes was detected by using CCK-8. The results indicated that hBMMSCs highly expressed B7-H1 molecule, hBMMSCs effectively inhibited the proliferation of T lymphocytes with a dose-dependent manner, and the inhibitory proliferation of T lymphocytes by hBMMSCs could be partially restored when the anti-B7-H1 mAb was used to block the B7-H1, the inhibitory rate of T lymphocyte proliferation decreased from 64.1% to 38.75%. It is concluded that B7-H1 highly expresses on hBMMSCs, the B7-H1 mediated signaling pathway (B7-H1/PD-1) involves in the mechanisms for hBMMSCs-mediated immunomodulation.

FLT3-ITD detection of free DNA in plasma from 235 patients with acute myeloid leukemia and its clinical significance / 中国实验血液学杂志

This study was purposed to evaluate the clinical significance of FLT3-ITD of free DNA in plasma from patients with AML. Free DNA in plasma of 235 patients with AML were extracted and identified by globin gene. FLT3 was amplified by PCR and compared with detected results of leukemic cellular DNA (BM or PB). The results indicated that out of total 235 patients, globin gene in plasma free DNA was successfully amplified from 190 cases. In 188 newly diagnosed, replaced and refractory cases, 35 cases showed ITD mutation (19%). And they also showed ITD mutation in leukemic cellular DNA. But in 47 patients in remission, 2 patients with FLT3-ITD mutation of free DNA in plasma had no mutation in cellular DNA, but got relapse early. Compared with patients of FLT3-wt, patients with FLT3-ITD mutation had increased WBC count and expression rate of CD7, CD56 and decreased CR rate. It is concluded that leukemic-specific DNA in plasma can be detected in AML patients and consistent with detected results of leukemic cellular DNA. Furthermore, the free DNA in plasma is more sensitive for MRD monitoring in remitted patients. FLT3-ITD detection plays an important role in evaluation of prognosis and molecular target therapy for AML patients.

Influence of hOCT1 polymorphism on imatinib mesylate effectiveness in chronic myelogenous leukemia patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the correlation between hOCT1 polymorphism and imatinib mesylate (IM) effectiveness in chronic myelogenous leukemia(CML) patients, and to provide for the clinical individual personalized therapy.</p><p><b>METHODS</b>Fifty-three CML and 23 non-CML patients were enrolled in this study. Blood or bone marrow samples were collected. Amplification refractory mutation system (ARMS)-polymerase chain reaction was used to amplify the polymorphisms gene segment of hOCT1-P283L, R287G and M408V and their frequencies were statistically analysed. With clinical outcomes, the correlation between hOCT1 polymorphism and IM effectiveness in CML was analyzed.</p><p><b>RESULTS</b>(1) For 74 Han Chinese, the allele frequencies of hOCT1-P283L, R287G and M408V were 39.86%, 29.05% and 45.27%, respectively. (2) The genotypes of hOCT1-P283L, R287G and M408V in 2 Tibetan Chinese were CC, CC, AG and CC, CG, AG, respectively. (3) In the CML patients with IM optimal response, the frequencies of 283T and 287G allele were predominant (P<0.05). No significant difference was found in the frequency distribution of hOCT1-M408V genotype and allele among the 3 different response groups (P>0.05).</p><p><b>CONCLUSION</b>(1) Three single nucleotide polymorphisms (cSNP) P283L, R287G and M408V were found in the hOCT1 gene from 76 Chinese. (2) hOCT1 gene polymorphism is associated with the long-term molecular response of CML patients received IM therapy, indicating that the polymorphisms of hOCT1-283T, 287G may be good predictors for IM response. (3) There is no correlation between the polymorphisms of hOCT1-P283L, R287G, M408V and secondary IM resistance in CML patients.</p>

Effect of 1,4-benzoquinone on growth of hematopoietic myeloid progenitor cells with IFN-gamma different genotypes / 中国实验血液学杂志

This study was aimed to investigate the effect of 1,4-benzoquinone (1,4-BQ) on growth of myeloid progenitor cells with IFN-gamma different genotypes and to compare its differences. Polymerase chain reaction (PCR) was used to amplify the polymorphism gene segment of IFN-gamma +874 A/T in 36 cord blood (CB) specimens. The specimens were divided into three groups (AA, AT and TT group). MNCs were planted on complete methylcellulose medium containing different concentrations of 1,4-BQ. The colony-forming units (CFU) were assayed, the differences of colony growth in specimens with different genotypes (AA, AT and TT) under 1,4-BQ exposure were analyzed. The results showed that frequencies of AA, AT and TT genotypes were 5.56%, 88.89% and 5.56% in the 36 CB samples respectively. Comparing colony numbers of IFN-gamma +874 AA, AT and TT genotype indicated that there was significant difference (p(AA) = 0.033, p(AT) = 0.009, p(TT) = 0.001, < 0.05). Significant cytotoxicity was observed after exposure to concentrations of 1,4-BQ > or = 5 micromol/L. Cytotoxic response of 1,4-BQ was dose-dependent. Under the same concentration of 1,4-BQ, there were no significant differences in capacity of cell colony growth between 3 groups (AA, AT and TT). Colony numbers of specimen No 3 in AT group and specimen No 2 in TT group were less than those of other specimens significantly. It is concluded that the hematopoietic myeloid progenitor cells cultured in the presence of 1,4-BQ show a dose-dependent cytotoxic response, but there are no significant differences in colony growth of IFN-gamma different genotypes (AA, AT and TT) under the same concentration of 1,4-BQ.

Single nucleotide polymorphism of interferon-gamma gene +874 T/A in patients with aplastic anemia / 中国实验血液学杂志

The aim of this study was to investigate whether the single nucleotide polymorphism (SNP) of interferon-gamma (IFN-gamma) +874 T/A correlates with aplastic anemia. Amplification refractory mutation system-polymerase chain reaction was used to amplify the polymorphism gene segment of IFN-gamma +874 A/T from 54 aplastic anemia patients and 51 healthy adults. The results showed that the frequency of IFN-gamma +874 TT genotype and T allele was significantly higher in patients with aplastic anemia than that in the healthy adults (42.6% vs 17.6%, chi2=13.780, p=0.01; 65.7% vs 39.2%; chi2=14.811, p<0.001). In conclusion, IFN-gamma +874 A/T gene polymorphism is correlated with the susceptibility of aplastic anemia, but not significantly correlated with the the severity of aplastic anemia.

Expression of tumor suppressor gene pten in patients with myelodysplastic syndrome and acute myeloid leukemia / 中国实验血液学杂志

To study the expression and significance of pten gene in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), RT-PCR and Western blot were respectively applied to detect pten mRNA and PTEN protein in Jurkat cells (as negative control), in bone marrow nucleated cells of 35 patients with MDS, 45 patients with AML and 20 normal control. The results showed that pten mRNA expression could not be detected in Jurkat cells, and the positive rate in MDS patients (77.1%) was significantly lower than that in normal control group (90.0%) (p > 0.05), while significant difference was found between AML patients and normal control (60.0% vs 90.0%, p < 0.05); the positive rate in MDS-RAEB patients (70.0%) was lower than that in MDS-RCMD (86.7%); positive rate in de novo and relapsed AML patients (53.3%) was lower than that in AML patients in CR (73.3%), but statistics tests did not show significant difference (p > 0.05). The results of relative expression level of pten mRNA in all groups indicated that both relative expression levels in MDS patients and AML patients were definitely lower than that in normal control group (p < 0.005); the relative expression level in MDS-RAEB patients was lower than that in MDS-RCMD patients (p < 0.05); and in de novo and relapsed AML patients was obviously lower than that in AML patients in CR (p < 0.001). However, there was no significant difference between MDS and AML patients (p > 0.05). The positive rate of PTEN protein expression in both MDS (65.7%) and AML (54.8%) patients were lower than that in normal control (90.0%) (p < 0.05), and there was no significant difference when comparing MDS-RCMD patients (80.0%) with MDS-RAEB patients (55.0%) (p > 0.05), but positive rate of PTEN protein expression in de novo and relapsed AML patients (44.4%) was significantly lower than that in AML patients in CR (73.3%) (p < 0.05). It is concluded that the complete loss of pten mRNA in MDS and AML is uncommon, but the relative expression level in both diseases is significantly lower than that in normal people. The positive rates of PTEN protein expression in both MDS and AML patients are lower, compared with normal people, but are not in accordance with the expression of pten mRNA. The abnormalities of pten gene expression may be involved in the pathogenesis of MDS and AML.

The clinical significance of Ig heavy chain and TCR gamma gene rearrangement detected in free DNA in plasma in patients with non-Hodgkin lymphoma / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the clinical significance of IgH and TCR gamma gene rearrangement in plasma free DNA in patients with non-Hodgkin Lymphoma (NHL).</p><p><b>METHODS</b>Plasma free DNA in 74 patients with NHL were extracted and identified by Globin gene. IgH (FR3A/VLJH), TCR gamma (TVG/TJX) clonal rearrangements were amplified by PCR and compared with results of mononuclear cell DNA and pathological biopsy sample DNA.</p><p><b>RESULTS</b>Plasma free DNAs were successfully obtained from 58 cases (35 B-NHL and 23 T-NHL) of newly diagnostic, refractory and relapsed NHL out of total 74 patients (78.4%), but not found in the rest 16 patients in remission. Of 35 B-NHL cases, 31 showed IgH rearrangement (88.6%), and none with TCR gamma rearrangement; of 23 T-NHL cases, 8 showed TCR gamma rearrangement (34.8%), and 2 with IgH gene rearrangement synchronously. In comparison with the results of IgH and TCR gamma gene rearrangement in biopsy samples in 30 B-NHL cases, 26 cases in plasma free DNA (86.7%) and 24 in biopsy samples (80%) were positive (P > 0.05). In 20 T-NHL patients, 7 cases in plasma cell-free DNA (35%) and 6 cases in biopsy samples (30%) were positive (P >0.05).</p><p><b>CONCLUSIONS</b>Tumor-derived DNA could be detected in plasma from underlying cancer patients. For NHL patients, detecting IgH and TCR gamma gene rearrangement in plasma free DNA has the same clinical significance as in biopsy samples.</p>

Study of molecular mechanism of tanshinone II A inducing differentiation in acute promyelocytic leukemia NB4 cells / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate molecular mechanism of tanshinone II A inducing differentiation and apoptosis in acute promyelocytic leukemia NB4 cells.</p><p><b>METHOD</b>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.</p><p><b>RESULT</b>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.</p><p><b>CONCLUSION</b>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.</p>

Mutation of mitochondria cytochrome oxidase gene in patients with myelodysplastic syndrome / 中国实验血液学杂志

The relationship between mitochondria gene mutation and hematological malignancies has been focusing on as a key point in recent studies. This study was aimed to investigate whether in patients with myelodysplastic syndrome (MDS) exists mitochoudria cytochrome oxidase COI and COII gene mutations different from normal tissues and to analyze whether these mutations are "hot spot" mutations. Eighteen MDS patients aged from 20 to 70 years old were brought into this study, including 2 of RA, 3 of RCMD, 7 of RAEB, 5 of AML (transformation from MDS), and 1 of MDS/MPD. The total DNA was extracted both from bone marrow cells and buccal cells of the same patients. A pair of primers was designed to amplify a fragment with 528 base pair (7181 - 7709) by PCR technique, which contained high frequency mutation area of cytochrome oxidase COI and COII gene based on the literature reports. The PCR products were purified and sequenced as bidirection to confirm if there is any mutation. The results of sequence of COI and COII gene from MDS patient bone marrow cells were compared with both the standard sequence from GenBank and the sequence from MDS patient buccal cells. The results showed that 3 single nucleotide changes in 528 bp cytochrome oxidase gene fragment from 18 MDS patients were confirmed. They were 7674 T-->C, 7353 A-->G, and an insert mutation of G at 7702. The former two mutations caused isoleucine-->methionine, and methionine-->viline. The 7702G ins was only confirmed with marrow cells in a patient, and caused a frame shift, which suggested that the mutation might be related to MDS cells. It is concluded that some of "hot spots" of mtDNA mutation in cytochrome oxidase (COI, COII) gene from our MDS patients are failed to be confirmed, but 3 new mutations on this gene are found, which suggested that mitochondria DNA mutations in MDS patients still have much complexity and heterogeneity, mtDNA mutation may be a prophase or an accompany phenomenon of this disease.

Role of gammadeltaT cells in pathogenesis of acquired pure red cell aplastic anemia / 中国实验血液学杂志

This study was purposed to investigate the changes in quantum and function of gammadelta T cell subsets, and to explore its significance in pathogenesis of acquired pure red cell aplastic anemia (A-PRCA). Eleven patients were diagnosed as A-PRCA based on bone marrow smear and biopsy, and were treated with cyclosporine A and glucosidorum tripterygll totorum. The flow cytometry technique was used for analyses of T cells subsets and gammadelta T cells. Furthermore, peripheral mononuclear cells (MNC) isolated from A-PRCA patients were cultured in RPMI 1640 medium (10(5) cells/ml) containing 10% FCS, phytohemagglutinin (PHA, 10 microg/ml), and recombinant human interleukin-2 (rIL-2, 50 U/ml) for two weeks, then gammadelta T cells were isolated with the TCRgammadelta Microbead Kit from cultured cells. The collected gammadelta T cells were incubated with normal control bone marrow MNC in RPMI 1640 medium (37 degrees C, 5% CO2 atmosphere) for CFU-E, CFU-GM, and BFU-E colony assay. The result showed that compared with the control group, CD3(+), CD8(+) cells increased significantly in the patient group (P < 0.05), the CD4(+)/CD8(+) ratio decreased and reversed, and gammadelta T cells were significantly increased in patient group (P < 0.05). After treatment with cyclosporine A, 9 out of 11 patients got good response, and CD3(+), CD8(+) cells in the responding patient decreased, the ratio of CD4(+)/CD8(+) returned to normal, and gammadelta T cells also decreased to normal range. Moreover, in vitro culture, the gammadelta T cells isolated from A-PRCA patients showed an inhibiting action to CFU-E and BFU-E but not to CFU-GM in a dose-dependent manner. It is concluded that gammadelta T cells increase in A-PRCA patients, and decrease in parallel to normal range with significant improvement of anemia symptoms after immune suppressive therapy. The gammadelta T cells isolated from A-PRCA patients showed an inhibiting action to CFU-E and BFU-E but not to CFU-GM in vitro culture, suggesting that gammadelta T cells may bring an impact on the research of A-PRCA pathogenesis. Cyclosporine A demonstrated better therapeutic effect on A-PRCA patients.

Effect of arsenic trioxide on bone marrow stromal cells of patients with multiple myeloma / 中国实验血液学杂志

To explore the effect of arsenic trioxide (As2O3) on growth and secretion of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) of bone marrow stroma cells (BMSC) from the patients with multiple myeloma (MM). Specimens of bone marrow aspiration from MM patients were used to establish BMSC cultures. BMSC and human MM cell line CZ-1 were cultured together or alone in the absence or presence of As2O3 at various concentrations (1-20.0 micromol/L). Cell growth inhibition was assessed by MTT assay, cytokines in the culture supernatants were measured with ELISA. The results showed that As2O3 had cytostatic effect on CZ-1 with fifty percent growth inhibition (IC50) for 48 hours at 2.3 micromol/L. As2O3 did not inhibit the growth of BMSC. High levels of IL-6 and VEGF have been found in the culture supernatants of BMSC from MM patients. Cytokine production of BMSC treated with As2O3 significantly decreased as compared with controls (P < 0.05). Excitingly, even the increased cytokine production triggered by adhesion of MM cell and BMSC was also inhibited by As2O3. It is concluded that As2O3 has no inhibitory effect on cell growth of BMSC, but inhibit the production of IL-6 and VEGF by BMSC.

Osteoblasts differentiated from human marrow bone mesenchymal stem cells support hematopoietic stem/progenitor cells from umbilical cord blood / 中国实验血液学杂志

This study was aimed to construct a two-dimensional culture system by using osteoblasts induced from human marrow mesenchymal stem cells (MSC) and to investigate its support effect on survival of hematopoietic stem/progenitor cells for umbilical cord blood (UCB) ex vivo. MSCs were isolated from adult human bone marrow and were cultured, the second generation of MSCs were induced into osteoblasts which were irradiated with 20 Gy gamma rays in a Cobalt 60 source and confluenced into a feeder layer. CD34(+) cells were selected from fresh umbilical cord blood samples by using Microbead Kit of MiniMACS and seeded into the two-dimensional culture system to culture ex vivo without exogenous cytokines. By using colony-forming assay, high proliferative potential colony-forming cell assay, and long-term culture initiating cell assay, the ability of the two-dimensional system to culture HSCs/HPCs was observed. The results showed that the osteoblasts induced from bone marrow MSC in constructed two-dimensional culture system displayed more significant support effect on survival of hematopoietic stem/progenitor cells from umbilical cord blood (UCB) ex vivo, compared with other culture systems, especially on long term HSCs survival ex vivo. It is concluded that the two-dimensional culture system constituted by osteoblasts induced from human MSCs has certain ability of supporting maintenance and multipotency of HSCs/HPCs from umbilical cord blood in vitro, especially sustaining survival of HSC in long-term culture. It has also been proved that osteoblasts play a crucial role in regulation of HSC growth.

Experimental study of donor natural killer cells reducing graft-versus-host disease and enhancing engraftment / 中国实验血液学杂志

The purpose of this study was to investigate whether pretransplant infusion of reactive natural killer cells (NK cells) from donor or recipient can reduce graft-versus-host disease (GVHD) and enhance engraftment in bone marrow transplantation (BMT). Recipient BALB/c mice were divided into 4 groups after received 6.5 Gy total-body irradiation (TBI): control group 1 was treated with nothing, control group 2 received BMT alone, experiment group 1 received BMT and autoreactive NK cells, experiment group 2 received BMT and alloreactive NK cells. Life span, clinical and pathologic changes of GVHD and chimerism rate of each group were evaluated. The results showed that all mice were survival in control group 1. The life span was shorter in experiment group 1 than that in control group 2 (P < 0.05) and longer in experiment group 2 than that in control group 2 (P < 0.01). GVHD was higher in score of experiment group 1 than in control group 2 (P < 0.05) but lower in experiment group 2 than that in control group 2 (P < 0.01). The donor chimerism rate in both two experiment groups were higher than that in control group 2 (P < 0.05), however, the donor chimerism rate was higher in experiment group 2 than that in experiment group 1 (P < 0.01). It is concluded that pretransplant infusion of alloreactive donor NK cells can prolong life span, reduce the degree of GVHD and enhance engraftment. But autoreactive recipient NK cells can shorten life span, aggravate the degree of GVHD and also enhance engraftment, which is weaker than that using alloreactive donor NK cells.

Immune regulatory effect of human bone marrow mesenchymal stem cells on T lymphocyte / 中国实验血液学杂志

To investigate the immune regulatory effects of human bone marrow mesenchymal stem cells on alloantigen T lymphocyte in vitro, human MSCs were isolated and expanded from bone marrow cells, and identified with cell morphology, and the phenotypes were assessed by immunohistochemistry and flow cytometry. As the stimulation factor of T lymphocytes proliferation, either PHA or dendritic cells isolated from cord blood were cocultured with CD2(+) T lymphocytes from peripheral blood mononuclear cells by magnetic beads with or without MSC in 96-well plats for seven days. T cell proliferation was assessed by [(3)H]-thymidine incorporation using a liquid scintillation counter. T cell subsets, Th1, Th2, Tc1 and Tc2 were analyzed by flow cytometry after co-culture of CD2(+) T cells with MSCs for 10 days. The results showed that a significant decrease of CD2(+) T cell proliferation was evident when MSC were added back to T cells stimulated by DC or PHA, and an increase of Th2 and Tc2 subsets were observed after co-culture of MSC with T lymphocytes. It is suggested that allogeneic MSC can suppress T cell proliferation in vitro and the cause of that was partly depend on interaction of cells and the alteration of T cell subsets.

Study on Growth and Maturation of Megakaryocyte Progenitors In Vitro in Patients with Chronic Idiopathic Thrombocytopenic Purpura / 中国实验血液学杂志

To investigate the growth and maturation of megakaryocyte progenitors in patients with chronic idiopathic thrombocytopenic purpura (CITP), plasma clot culture and GPIIIa monoclonal antibody and ABC immuno- histochemical kit were used to assay CFU-Meg and BFU-Meg, and the area and diameter of GPIIIa(+) cells were determined by image analyzer in 33 CITP cases. It was found that CFU-Meg and BFU-Meg were 39.27 +/- 21.44 and 5.62 +/- 3.93 per 2 x 10(5) MNC, respectively, in CITP patients, there were no significant differences with those in control group. While the area of GPIIIa(+) cells was (134.90 +/- 6.08) micro m(2) and diameter was (12.89 +/- 3.66) micro m, those were lower than those in control group. In patients with normal number of megakaryocytes on marrow smears, CFU-Meg and BFU-Meg were 19.43 +/- 7.28 and 4.67 +/- 1.53, respectively, the values were lower as compared to control group. The positive correlation was showed between the total megakaryocytic colonies and the number of megakaryocytes on marrow smears, r = 0.6503, and there was no correlation with blood platelet counts and course of disease. The results suggest that there was a maturation disturbance of megakaryocyte progenitor in CITP patients and lower proliferative potential in patients with normal megakaryocyte counts on marrow smears.