Effect of Hemoglobin on Efficacy of CAR-T Therapy in Patients with Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of hemoglobin (Hb) on the efficacy of chimeric antigen receptor T cell therapy (CAR-T) in patients with multiple myeloma (MM).@*METHODS@#From June 2017 to December 2020, 76 MM patients who received CAR-T therapy in the Department of Hematology, The Affiliated Hospital of Xuzhou Medical University, with complete clinical data and evaluable efficacy, were selected as the research objects. According to the receiver operating characteristic (ROC) curve, the best cut-off value was obtained. The patients were divided into groups on the basis of Hb 105.5 g/L as the cut-off value. The age, sex, serum calcium, β2-microglobulin, serum creatinine, lactate dehydrogenase (LDH), and the influencing factors of CAR-T treatment efficacy in MM patients were analyzed.@*RESULTS@#Hb was an influencing factor of efficacy. Univariate analysis showed that Hb, LDH, and albumin affected the efficacy of CAR-T therapy. Multivariate analysis showed that Hb ( OR=1.039, 95% CI: 1.002-1.078) and LDH ( OR=1.014, 95% CI: 1.000-1.027) were the influencing factors for the efficacy of CAR-T therapy.@*CONCLUSION@#The efficacy of CAR-T therapy in MM patients with low Hb is poor, and Hb is a factor affecting the efficacy of CAR-T therapy.

Observation of liver indexes in patients with relapsed/refractory multiple myeloma treated with CAR-T-cells based on BCMA / 中华血液学杂志

Objective: To observe the characteristics of the evolution of liver indexes in patients with relapsed/refractory multiple myeloma (RRMM) treated with CAR-T-cells based on BCMA. Methods: Retrospective analysis was performed of patients with RRMM who received an infusion of anti-BCMA CAR-T-cells and anti-BCMA combined with anti-CD19 CAR-T-cells at our center between June 1, 2019, and February 28, 2023. Clinical data were collected to observe the characteristics of changes in liver indexes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and direct bilirubin (DBIL) in patients, and its relationship with cytokine-release syndrome (CRS) . Results: Ninety-two patients were included in the analysis, including 41 patients (44.6%) in the group receiving a single infusion of anti-BCMA CAR-T-cells, and 51 patients (55.4%) in the group receiving an infusion of anti-BCMA combined with anti-CD19 CAR-T-cells. After infusing CAR-T-cells, 31 patients (33.7%) experienced changes in liver indexes at or above grade 2, which included 20 patients (21.7%) with changes in one index, five patients (5.4%) with changes in two indexes, and six patients (6.5%) with changes in three or more indexes. The median time of peak values of ALT and AST were d17 and d14, respectively, and the median duration of exceeding grade 2 was 5.0 and 3.5 days, respectively. The median time of peak values of TBIL and DBIL was on d19 and d21, respectively, and the median duration of exceeding grade 2 was 4.0 days, respectively. The median time of onset of CRS was d8, and the peak time of fever was d9. The ALT, AST, and TBIL of patients with CRS were higher than those of patients without CRS (P=0.011, 0.002, and 0.015, respectively). CRS is an independent factor that affects ALT and TBIL levels (OR=19.668, 95% CI 18.959-20.173, P=0.001). The evolution of liver indexes can be reversed through anti-CRS and liver-protection treatments, and no patient died of liver injury. Conclusions: In BCMA-based CAR-T-cell therapy for RRMM, CRS is an important factor causing the evolution of liver indexes. The evolution of liver indexes after CAR-T-cell infusion is transient and reversible after treatment.

Expression and Significance of PD-1, TIM-3 and VISTA on T Cell of Acute Myeloid Leukemia Patients / 中国实验血液学杂志

OBJECTIVE@#To study the expression of multiple negative costimulatory molecules on peripheral blood T cells in patients with acute myeloid leukemia (AML) and its affection on prognosis.@*METHODS@#The peripheral blood samples from patients with newly diagnosed AML, complete remission (CR), and no-remission (NR) were collected, the expression levels PD-1、VISTA and TIM-3 in CD4 and CD8 T cells were detected by flow cytometry , and the clinical data of patients were analyzed.@*RESULTS@#The expression levels of PD-1、VISTA and TIM-3 of CD4 and CD8 T cells in the newly diagnosed AML patients were significantly higher than those in control group (P＜0.05). The expression levels of PD-1、TIM-3 and VISTA of CD4 and CD8 T cells in the CR group were significantly lower than those in newly diagnosed and the NR group (P＜0.05). The TIM-3 expression level positively correlated with VISTA expression level of CD4 and CD8 T cells in newly diagnosed AML patients (r=0.85 and 0.73). The VISTA and PD-1 expression level of CD4 T cells in newly diagnosed AML, NR after first induction chemotherapy and high risk patients significantly increased (P＜0.05), the TIM-3 expression level of CD8 T cells in high risk group significantly increased (P＜0.05), and the VISTA expression level of CD8 T cells in CBFβ-MYH11 mutation-positive group significantly decreased (P＜0.05).@*CONCLUSION@#The expression of PD-1、TIM-3 and VISTA in AML peripheral blood T cells may be involved in the immune escape of AML and can be the targets of treatment for acute myeloid leukemia patients.

Curative Efficacy of Rituximab for ITP Patients with Different Sensitivity to Hormone / 中国实验血液学杂志

OBJECTIVE@#To investigatc the curative efficacy of low dose rituximab for glucocorticoid ineffective on dependent ITP patients and its relation with sensitivity to glucocorticoid so as to provide reference basis for rational use of drugs in clinical treatmant.@*METHODS@#Seventy-ninth ITP patients enrolled in this study included the glucocorticoid-ineffective patients (19 cases) and glucocorticoid-dependent patients (60 cases). All ITP patients were treated with regimen consisted of high dose dexamethasone plus low dose rituximab (dexal-methasone 40 mg/d for 4 days per os, ritaximab 100 mg by intravenous infusion at D7, 14, 21 and 28 respectively). The patients after treatment were followed-up for 12 month, and the relation of patients sensitivity to glucocorticoid with therapentic response of rituximab was analyzed. The changes of Treg cell ratio and BAFF, IL-2 and sCD40L levels before and after treatment were detected by flow cytometry and ELISA respectively.@*RESULTS@#The overall response rate (ORR) of patients treated with above- mentioned regemen at 1, 3, 6 and 12 months after treatment was 79.7% (63/79), 69.6% (55/79), 63.3% (50/79) and 60.8% (48/79) respectivcly, out of which the ORR of glucocorticoid ineffective and glucocorticoid-dependent ITP patients treated with above-mentioned regimen at 1, 3, 6 and 12 months after treatment was 47.4% (9/19) vs 90.0% (54/60), 36.8% (7/19) vs 80.0% (48/60), 21.1% (4/19) vs 76.7% (46/60), 21.1% (4/19) vs 73.3% (44/60), and the difference between 2 groups was statistically significant. The detection of T reg cell showed that the T reg cell ratio in glucocorticoid- ineffective and dependent patients at 1, 3, 6 and 12 months after treatment was (1.70±0.43)% vs (3.47±0.72)%, (1.66±0.33)% vs (4.29±0.91)%, (1.71±0.37)% vs (4.44±0.97)%, (3.36±0.54)% vs (4.29±1.04)%, respectively. The detection of cytokines showed that the levels of BAFF, IL-2 and sCD40L in plasma of glucocorticoid-dependent patients at 1 month after treatment significanlly decreased (P＜0.05), the levels of BAFF, IL-2 and sCD40L in plasma of glucocorticoid-ineffective patients although decreased at 1 mouth after treatment, but there was no statistical difference as compared with glucocosticoid-depenment patients.@*CONCLUSION@#The treatment of glucocorticoid-dependent ITP patients with rituximab is more effective. The regulatory effect of rituximab on the T-reg cells, BAFF, IL-2 and sCD40L may be one of its mechanisms.

Multivariate Analysis of Factors Influencing Recovery from Hemorrhagic Cystitis after Allo-HSCT / 中国实验血液学杂志

OBJECTIVE@#To analyze the incidence of hemorrhagic cystitis (HC) after allogeneic hematopoietic stem cell transplantation and the factors affecting HC, so as to provide clinical evidence for further treatment of HC.@*METHODS@#The HC of 113 patients after allogeneic hematopoietic stem cell transplantation in Affiliated Hospital of Xuzhou Medical University between the years 2014-2016 was analyzed respectively. All cases of HC were divided into HC group and non-HC(control) group. The follow-up time: from preeonditionig day to 180 d after transplantation. The 10 clinical parameters were selected for univariate analysis with COX regression analysis: sex, age (＜25 years and 25 years), primary disease, conditioning regimen with anti-thymoglobulin(ATG), sex-mismatch in recipients, haploidential HSCT, cytomegalovirus (CMV) viremia, EB viremia, graft-versus-host disease (GVHD), and primary disease relapse, the factors significant at the 0.1 level in univariate analysis should be further evaluated by multivariate analysis using a COX regression analysis. The difference was significant at P＜0.05 in multivariate analysis.@*RESULTS@#The HC occured in 31 of 113 patients (27.4%), with 5 cases of grade I (5.5%), 19 of grade II (16.8%), 5 of grade III (4.4%), and 2 of grade IV (1.8%). The median time of HC onset was 37 days (26-70 d) after transplantation. The median duration of HC was 14 days (5-55d). Univariate analysis showed that conditioning with anti-thymoglobulin (ATG) (RR=6.170, 95%CI: 1.875-20.306, P＜0.01), CMV viremia (RR=7.633, 95%CI：2.318-25.133) (P＜0.01), haploidentical HSCT (RR=0.307, 95%CI：0.137-0.686, P＜0.01), GVHD (RR=1.891, 95%CI：0.918-3.898, P＞0.05) were the risk factors for recovery from HC. The multivatiate analysis of above-mentioned risk factors with statistical significance showed that only CMV viremia (RR=4.770, 95%CI: 1.394-16.326, P＜0.05) was the indentified risk factor affecting the recovery from HC.@*CONCLUSION@#Monitoring CMV viremia and antivirotic treatment are effective measurs to prevent the occurrence of HC and promote the recovery from HC.

Role of Ca-NFAT Signaling Pathway in Ph ALL Drug-resistance Mediated by Bone Marrow Stromal Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the role of Ca-NFAT signaling pathway in Ph-ALL drug resistance mediated by bone marrow stromal cells.@*METHODS@#The transcription level of NFAT mRNA in Sup-B15 cells and Ph ALL primary cells was detected by polymerase chain reaction. The expression of P-glycoprotein in Sup-B15 cells was detected by flow cytometry. The change of NFAT protein in Sup-B15 cells was detected by Western blot. AnnexinV/7-AAD was used to label cells. Flow cytometry was used to detect cell apoptosis; Fluo 3-AM dye was used to label cells, and flow cytometry used to detect changes of Ca concentration in leukemia cells.@*RESULTS@#NFAT expression could be detected in both Sup-B15 and Ph ALL primary cells; P-glycoprotein could not be detected by flow cytometry; CAS could significantly inhibit NFAT protein expression in clinically applied drug concentrations (2.5, 5 μmol/L); Clinically applied concentration of CAS (2.5, 5 μmol / L) has no significant effect on the apoptosis of Sup-B15 cells, while higher concentration of CAS (10 μmol / L) could induce apoptosis of Sup-B15 cells. Bone marrow stromal cells OP9 could, decrease the sensitivity of Sup-B15 cells and Ph ALL primary cells to imatinib (IM); After co-culture with bone were marrow stromal cells, the Ca concentration in Sup-B15 cells was enhanced, the levels of NFAT protein and nullear protein in sup-B15 cells also were enhanced. The addition of CAS in co-culture system could inlibit the Ca-NFAT signaling pathway, reduce the protective effect of OP9 on Sup-B15 cells.Conclution:The Ca-NFAT sigualing pathway, contributes to the survival of Ph ALL cells. Bone marrow stromal cells can mediate the resistance of Ph ALL cells to IM by activating Ca-NFAT signaling pathway.

Effect of Stably Down-regulating FMI Expression of K562 Cells on Sensitivity of K562 cells to Imatinib Mesylate / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of stably down-regulating the FMI expression of K562 cells on the sensitivity of K562 cells to Imatinib (IM) and its possible mechanism.@*METHODS@#Western-blot was used to detect the expression of FMI protein in K562 cells and peripheral blood mononuclear cells from the patients with chronic myelogenous leukemia, chronic myeloid blast crisis and healthy volunteers. The specific interference sequences targeting at the human FMI gene were designed and ligated into the lentiviral vector LV3; the three plasmid system-packaged lentivirus particles were used to transfect K562 cells to screen K562 cells that stably down-regulated FMI. CCK-8 assay and flow cytometry were used to determine effect of IM on cell proliferation and apoptosis. The transcription level of FMI and Fz8 in leukemia cells was detected by fluorescent quantitative PCR. The protein expression levels of FMI, Fz8, NFAT1, BCR-ABL and β-catenin in leukemia cells were detected by Western-blot.@*RESULTS@#The expression of FMI protein could be detected in peripheral blood mononuclear cells of the patients with CML-BC and K562 cells, the FMI expression could not be detected in all the patients with CML-CP and healthy volunteers. The recombinant lentiviral vector LV3/FMI had been successfully constructed the lentivirus was packaged, and the K562 cells stably down-regulating the FMI protein were screened. After stable down-regulation of FMI expression in K562 cells, the proliferation rate of leukemia cells decreased and the apoptosis rate was increased under the same drug concentration. Both the transcription and protein expression levels of Fz8 decreased. The NFAT1 total protein level increased, as well as the nuclear translocation of protein was enhanced. There was no significant change in the expression level of BCR-ABL fusion protein. The expression level of β-catenin protein decreased.@*CONCLUSION@#After the stable down-regulation of FMI expression, the sensitivity of K562 cells to IM and apoptosis of cells increase, which are performed possibly by inhibiting the FMI-Fz8 signaling pathway and activating the Ca-NFAT and Wnt/β-catenin signaling pathway.

Prognostic Significance of CD45CD117 Cells in Patients with Acute Myeloid Leukemia after Complete Remission / 中国实验血液学杂志

OBJECTIVE@#To investigate the predictive value of CD45CD117 phenotype-abnormal cells (hereinafter referred to as "abnormal cells") for relapse and prognosis in adult patients with acute myeloid leukemia (AML) within 2 weeks after the first complete remission (CR1).@*METHODS@#The clinical data of patients with newly diagnosed AML (non-acute promyelocytic leukemia) admitted in our department from July 1, 2014 to June 30, 2017 were analyzed retrospectively, and the relationship between clinical features at the initial diagnosis and the abnormal phenotype cells of CD45CD117 within 2 weeks after CR1 with the prognosis were analyzed.@*RESULTS@#A total of 91 patients with CD45CD117 abnormal cells were detected. The median age was 51 years old, the median WBC count was 11.60×109/L, and the median ratio of bone marrow blast cells was 0.35 at initial diagnosis. According to the FAB classification, 1 (1.1%), 7 (7.7%), 38 (41.7%), 20 (22.0%), 21 (23.1%) and 4 (4.4%) patients were classifice as M0, M1, M2, M4, M5, and M6, respectively. According to the NCCN risk stratification, 30 (33.0%), 51 (56.0%), and 10 (11.0%) patients were determined as good, moderate, and poor prognosis, respectively. The median ratio of abnormal cells within 2 weeks after CR1 was 1.8500 (0.0236-8.0000)%. The median time from initiation of induction therapy to the acquisition of CR was 46 days, median recurrence-free survival time was 319 days, and median overall survival time was 352 days. A total of 45 patients relapsed, of which 14 died; 46 patients did not relapse, of which 3 died. The cutoff of abnormal cells by receiver operating characteristic curve (ROC) analysis was 2.055% (Se=0.733，Sp=0.761). The abnormal cell ratio was＞2.055% in 44 patients, the median ratio of abnormal cells was 3.075%, among which 33 patients relapsed and 12 patients died; the abnormal cell ratio was ＜2.055% in 47 patients, the median ratio of abnormal cells was 1.150%, 12 patients relapsed and 5 patients died. Regression analysis showed that WBC count＞50×10/L and abnormal cell ratio＞2.055% were independent risk factors for recurrence. The abnormal cell ratio＞2.055% group had a 2-year RFS rate of 54.3% and a 2-year OS rate of 52.8%. The abnormal cell ratio＜2.055% group had a 2-year RFS rate of 86.6% (P=0.018), and a 2-year OS rate of 85.3% (P＜0.05).@*CONCLUSION@#For adult AML patients, CD45CD117 phenotypical abnormal cells ratio＞2.055% within 2 weeks after CR1 is an independent risk factor for recurrence, which also is an dverse factor for RFS and OS.

Relationship between BCR-ABL and Prognosis and Affecting Factors for Prognosis in CML Patients Treated with TKI for 12 Months / 中国实验血液学杂志

@#<p><b>OBJECTIVE</b>To evaluate the long-term prognosis of CML patients whose BCR-ABL transcript level was warning and best response at 12 months of treatment with tyrosine kinase inhititor (TKI), and to investigate the factors affecting therapeutic efficacy and prognosis.</p><p><b>METHODS</b>The clinical data of patients with newly diagnosed CML were analyzed retrospectively. According to BCR-ABL transcript level, the 80 patients were divided into group A and group B, the patients with BCR-ABL >0.1% and ≤ 1% (warning response) were entolled in group A, and the patients with BCR-ABL ≤ 0.1% (best response) were enrolled in group B as control. The ratio of patients with main molecular response (MMR) and deep molecular response (DMR), as well as aquistation rate and cummulative rate of MR (DMR) at specified fine points in 2 groups were compared, the independent risk factors affecting the therapeutic efficacy and prognosis were analyzed.</p><p><b>RESULTS</b>The MMR and MR of the B group at 15, 18 and 24 months after TKI treatment were significantly higher than those of the A group, and the patients in the B group reached MR faster. In the 3 months, 6 months and 12 months after the demarcation point (TKI 12 months), the A group was much less easy to obtain MR (P<0.05) than the B group. Through survival analysis, there were more patients in the B group than the A group at different time points to reach MR, and the difference was statistically significant (P<0.01). The single factor analysis showed that the splenomegaly (below rib edge)> 10cm (P<0.01) and lactate dehydrogenase > 400 U/L (P<0.05) were the long-term warning factors for patients. Multivariate analysis showed that the size of the spleen was an independent factor (P<0.01) to affect the prognosis of the patients who had been warned for 12 months.</p><p><b>CONCLUSION</b>Patients at 12 months warning effect are slower and less easier to get DMR, which has a poor long-term prognosis. The size of the spleen in the patient at warning for 12 months of treatment effect can predict the relatively poor long-term prognosis. For a patient with a 12 months response to the warning, an early replacement therapy is available on the basis of combining other factors..</p>

Risk Factors and Prognosis of Hepatic cGVHD after Allogeneic Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze the risk factors and prognosis of hepatic chronic GVHD after allogeneic hematopoietic stem cell transplantation (allo-HSCT).</p><p><b>METHODS</b>The clinical data of 147 patients undergoing allo-HSCT from January 2013 to December 2016 were analyzed, the correlation between recipient age and sex, disease state, matched degree of HLA, donor sex, stem cell sources, ATG in GVHD prophylaxis, liver dysfunction during conditioning period, pre-transplant HBsAg, prior aGVHD and hepatic cGVHD were studied, and the correlation between hepatic cGVHD and prognosis were analysed.</p><p><b>RESULTS</b>Thirty-two patients had hepatic cGVHD, cumulative incidence of 26.4%. In univariate analysis, pre-transplant HBsAgand liver dysfunction during conditioning period were not significantly related with hepatic cGVHD (P>0.05). In multivariate analysis, prior acute GVHD (HR=2.087, P=0.045) was the independent risk factor for hepatic cGVHD, ATG (HR=0.231, P=0.000) was significantly related with a lower incidence of hepatic cGVHD. In univariate analysis, patients with hepatic cGVHD had a lower 2 years relapse rate (P=0.038).</p><p><b>CONCLUSION</b>Prior acute GVHD is the independent risk factor for hepatic cGVHD, the ATG can significantly reduce the incidence of hepatic cGVHD. Hepatic cGVHD has been found to relate with a lower 2 years relapse rate.</p>

Effect of Steadily Down-regulating VE-Cadherin Expression on Susceptibitity of K562 Cells to Chemotherapy / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of steadily down-regulating the expression of VE-cadherin on the chemotheraputic sensitivity of K562 cells, and explore its possible mechanism.</p><p><b>METHODS</b>Specifically targeting interference sequences carrying human VE-cadherin were designed, the recombinant lentiviral vector containing the IRES-GFP and NEO segment was constructed; recombinant lentivirus was generated by three-plasmids packing system, and transfected into K562 cells, then the cells steadily down-regulated were sorted. CCK-8 assay was performed to evaluate the VE-cadherin of chemotherapeutic (Imatinib) sensitivity of K562 cells. The apoptosis was analyzed by flow cytometry with Annexin V/7-AAD double labeling. The expressions of CD133 and ALDH1 mRNA were determined by real time PCR. The protein expressions of VE-cadherin, BCR-ABL and β-catenin were analyzed by Western blot.</p><p><b>RESULTS</b>The recombinant lentiviral vector pLB-shVEC-NEO-IRES-GFP was successfully constructed, packed into the lentivirus, then the K562 cells steadily down-regulating VE-cadherin expression was obtained. When VE-cadherin was down-rengulated in K562 cells, the proliferation rate was reduced while the the apoptosis rate was increased; the mRNA levels of CD133 and ALDH1 also were reduced; BCR-ABL fusion protein was not obviously changed; the total β-catenin protein, as well as the nuclear β-catenin protein were decreased in the K562/shVEC cells. Conclution: K562 cells are more susceptible to chemotherapy when VE-cadherin is down-regulated, that may be realized via reducing the stability and the nuclear transfer of β-catenin protein.</p>

A Modified Protocol for Rapid Establishing Mouse Model with Ph Positive Acute Lymphoblastic Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate a modified protocol of establishing mouse Ph ALL model so as to provide a more convenient and more powerful tool for Ph ALL studies.</p><p><b>METHODS</b>Immature B cells from BALB/c mice were transfected with the Mig190 retrovirus and infused into irradiated syngeneic mice. The immunophenotype was identified by flow cytometry, the BCR-ABL was identified by RT-PCR and Western blot. Leukemia cells isolated from sick mice were re-infused into syngeneic mice without irradiation.</p><p><b>RESULTS</b>The acute lymphoblastic leukemia was established in mice after Mig190 retroviral transfection, the lymphoblasts were observed by blood smears, the immunophenotype of these leukemic cells was CD19, and the BCR-ABL was detected by RT-PCR and Western blot, the immunophenotype was conformed as Ph ALL, the isolated leukemia cells infused into mice rapidly developed B-ALL.</p><p><b>CONCLUSION</b>A new protocol is established to generate mice with Ph ALL. This mouse model can provide a more simple and effective tool in comparison with the conversional protocols.</p>

Clinical Significance of Serum Thrombopoietin Levels in Patients with Aplastic Anaemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To evaluate the clinical significance of thrombopoietin (TPO) level in diagnosis of pateints with aplastic anaemia (AA).</p><p><b>METHODS</b>The TPO levels in sera from 54 AA patients and 119 healthy controls were examined. A total of 92 samples were collected from AA patients including 43 samples harvested at diagnosis, 23 samples in the cytopenic period after treatment, and 26 samples of patients in partial (n=10) or complete remission (n=16) following immunosuppressive treatment. Serum TPO levels were assessed by a sandwich-antibody ELISA that utilized a polyclonal rabbit antiserum for both capture and signal.</p><p><b>RESULTS</b>Serum samples from normal donors revealed a mean TPO level of 95.3±54.0 pg/ml, the mean TPO levels in AA sera collected at diagnosis and before onset of treatment were 2728±1074 pg/ml (P<0.001) compared with normal controls; mean platelet count at that time: 27×10/L). Serum TPO levels of AA patients in partial or complete remission after immunosuppressive treatment were significantly lower than TPO levels at diagnosis (P<0.001). However, despite normal platelet counts (mean 167×10/L), TPO levels remained significantly elevated in complete remission (mean TPO 1009±6590 pg/ml (P<0.001) compared with normal controls. There was a significant negative correlation between serum TPO levels and platelet counts in AA patients (coefficient of correlation r=-0.70, P<0.001).</p><p><b>CONCLUSION</b>TPO levels are highly elevated in sera of patients with AA. Thrombopoietin did not return to normal levels in remission, indicating normal platelet count in remission of AA needs sustained high level of TPO.</p>

Clinical effect of sevelamer in hyperphosphatemia of chronic renal failure patients with maintenance hemodialysis / 药物评价研究

Objective To investigate the clinical effect of sevelamer in hyperphosphatemia of chronic renal failure patients with maintenance hemodialysis.Methods Chronic renal failure patients (80 cases) with maintenance hemodialysis and hyperphosphatemia m The First People's Hospital of Nanyang from July 2013 to August 2016 were selected and divided into observation group and control group,40 cases in each group.Patients in control group were treated with calcium acetate,and patients in observation group were treated with sevelamer.The effectiveness indexes (serum phosphorus,serum calcium,calcium-phosphorus product and iPTH),clinical effect,interleukin (IL) 6,tumor necrosis factor (TNF)-α,hypersensitive c-reactive protein (hs-CRP),and adverse reaction before and after treatment were compared.Results After treatment,the serum phosphorus and calcium-phosphorus product of two groups were significantly lower than before treatment (P ＜ 0.05),and the observation group were significantly lower than control group (P ＜ 0.05).After treatment,the total effective rate of the observation group was 92.50％,significantly higher than 75％ of the control group,and the difference was statistically significant (P ＜ 0.05).The levels of IL-6 and TNF-αt in two groups and hs-CRP level in observation group were significantly lower than those before treatment (P ＜ 0.05);The observation group was significantly lower than control group,and the difference between two groups was statistically significant (P ＜ 0.05).The difference in the adverse reaction rate had no significant between two groups.Conclusion Sevelamer Carbonate Tablets have remarkable clinical effect in hyperphosphatemia of chronic renal failure patients with maintenance hemodialysis,and can effectively reduce the serum levels of inflammatory cytokines,which is safe and reliable.

Effect of BRD4 Inhibitor GSK525762A on Proliferation and Apoptosis of SUP-B15 Cells In Vitro and Its Possible Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of BRD4 inhibitor GSK525762A on the proliferation and apoptosis of Philadelphia chromosome-positive acute lymphoblastic leukemia SUP-B15 cells and its mechanism.</p><p><b>METHODS</b>SUP-B15 cells were treated with different concentration of GSK525762A, the proliferation-inhibition curve was assayed and plotted by CCK-8 method, the cell viability and apoptosis were detected by flow cytometry with Annexin V and 7-AAD staining. The transcripts of anti-apoptotic genes C-MYC, CDK6 and BCL-2 were detected by real-time PCR.</p><p><b>RESULTS</b>GSK525762A could inhibit significantly SUP-B15 cell proliteration in dose-and time-dependent manner; GSK525762A treatment could induce apoptosis of SUP-B15 cells. The levels of C-MYC,CDK6 and BCL-2 mRNA transcripts in SUP-B15 cells were reduced in GSK525762A-treated group.</p><p><b>CONCLUSION</b>The GSK525762A can remarkably inhibit proliferation and induce apoptosis of SUP-B15 cells. The down-regulation of apoptosis-related genes C-MYC,CDK6 and BCL-2 may be involved in the process of apoptosis.</p>

Effect of GRK6 on Proliferation of Multiple Myeloma MM1R Cells and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the regulatory effect of GRK6 on the proliferation of multiple myeloma cells and its mechanism.</p><p><b>METHODS</b>A lentivirus vector shRNA interfering in human GRK6 gene expression was constructed and trans-fected into multiple myeloma cells to obtain the cell line MM1R with stable down-regulation of GRK6 gene expression. The real-time quantitative PCR and Western blot were used to confirm the effectiveness of the GRK6 gene expression down-regulation mediated by lentivirus vector. The MM1R cells with most obvious down-regulation were selected to detect the effect of GRK6 gene on cell proliferation.</p><p><b>RESULTS</b>The lentivirus vector GRK6-shRNA interfering in human GRK6 gene was constructed succesufully and transfected into multiple myeloma cells, thereby the MM1R cell line with stable down-regulation of GRK6 gene was obtained. The CCK-8 assay showed that the proliferative viability of MM1R cells in experimental group was significantly lower than that in control group (P<0.05); the flow cytometry showed that cells in experimental group were arrested in G/Gphase(P<0.05); the Western blot detection showed that the Cyclin D1 and CDK4 levels in experiment group obviously decreased as compared with control group.</p><p><b>CONCLUSION</b>A lentivirus vector which can specifically interfere in GRK6 gene expression is constructed successfully, The MM1R cell line with stable down-regulation of GRK6 expression is obtained by transfection and screening. The down-regulation of GRK6 expression can arrest MM1R cells in G/Gphase, moreover inhibits the proliferation of MM1R cells by inhibition of Cyclin D1 and CDK4 levels.</p>

Establishment and Identification of Mouse Model of Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish a mouse model of Phacute lymphoblastic leukemia (ALL) for providing a valuable tool to facilitate the researches on PhALL.</p><p><b>METHODS</b>CML-like mice were generated by transfection to bone marrow cells of BABL/c with Mig190 retrovirus. The PhALL mouse model was established by infusion of sorted CML like mouse-derived BCR-ABLB cells into the mice of same linege. Immonophenotypes, BCR-ABL transcription and expression of these leukemic cells were detected by flow cytometry, RT-PCR and Western blot respectively.</p><p><b>RESULTS</b>CML-like presentation appeared in the mice after Mig190 virus transfection. After infusion with sorted BCR-ABLB cells, all the receipted mice eventually presented the clinical signs of acute leukemia. Flow cytometry analysis showed that these leukemic cells were positive for CD19; both RT-PCR and Western blot indicated that this mouse model is consistent with PhALL phenotypecally and molecalarlly.</p><p><b>CONCLUSION</b>A novel method to establish PhALL mouse model has been developed successfully, and this model can provide useful tool for the study of PhALL.</p>

Effect of ADAM10 Inhibitor GI254023X on Proliferation and Apoptosis of Multiple Myeloma H929 Cells and Its Possible Mechanisms / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of ADAM10 inhibitor GI254023X on the proliferation and apoptosis of multiple myeloma H929 cell line and its mechanisms.</p><p><b>METHODS</b>H929 cells were treated with different concentrations of GI254023X, the proliferation-inhibitive curve was assayed and plotted by CCK-8 method, the cell viability and apoptosis were detected by flow cytometry with Annexin V/7-AAD double staining. The cleavage of Notch1 protein (cleaved notch1) was determined by Western blot. The transcripts of Notch1 target gene Hes-1 were detected by real-time PCR.</p><p><b>RESULTS</b>The GI254023X inhibited the proliferation of H929 cells in the time- and dose- dependent manners. As compared with the control group, the apoptosis of cells increased along with enhancement of GI254023X concentration; The expression of cleaved Notch1 was down-regulated after the treatment with GI254023X. The levels of Hes-1 mRNA transcripts in H929 cells was reduced in GI254023X treated group.</p><p><b>CONCLUSION</b>GI254023X can remarkably inhibit the proliferation and induce the apoptosis of H929 cells. Its mechanism may be associated with inbihition of Notch1 activation.</p>

Effect of interfering the β-catenin by lentiviral vector-mediated RNAi on the biological behavior of mouse bone marrow mesenchymal stem cells / 中国实验血液学杂志

This study was aimed to investigate the effect of Wnt/β-catenin signaling pathway on the biologic behavior of mouse bone marrow mesenchymal stem cells (mBM-MSC) by constructing a RNAi lentiviral vector specific to β-catenin. Three pairs of shRNA coding sequences directed against different sites of β-catenin mRNA were designed and were linked into lentiviral vector plasmid PLB for constructing the PLB-β-catenin/shRNA1, PLB-β-catenin/shRNA2 and PLB-β-catenin/ shRNA3. Those plasmids and lentiviral packaging plasmids were co-transfected into the packaging cells 293FT, then the virus particles were collected and the viral titer was assayed after concentration, and these viral particles were infected to MSC. The flow cytometry was used to sort GFP (+) cells, Western blot and RT-PCR were used to verify the inhibitory effect of those cells on expression of β-catenin gene in cells, CCK-8 method was used to detect the cell proliferation level, Annexin-V/7-AAD was used to determine the cell apoptosis after interference, the cell scratch and transwell tests were used to detect the migration capability of MSC. The results showed that the efficient inhibition of β-catenin mRNA and protein expression, and the suppression of MSC proliferation were observed in group of PLB-β-catenin/shRNA2 (interference group), while there was no significant changes of MSC proliferation between negative group (PLB group) and the normal group (control group). The flow cytometric detection indicated that after induced by serum starvation for 72 h, the apoptosis of MSC increased in interference group, but there was no difference between PLB and control groups (P > 0.05). The cell scratch and transwell tests demonstrated that the migration capability of MSC in interference group decreased significantly, while the migration capability of MSC in control group was not changed obviously. It is concluded that the constructed specific RNAi lentivirus can effectively inhibit the expression of β-catenin gene, decrease expression level of β-catenin mRNA and protein. The Wnt/β-catenin signaling pathway plays an important role in biological behavior of BM-MSC.

Effect of VE-cadherin on sensitivity to Imatinib in Sup-B15 Philadelphia chromosome positive acute lymphoblastic leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the sensitivity of imatinib mesylate (IM) on Sup-B15 Ph⁺ acute lymphoblastic leukemia (ALL) cells knockdown of VE-cadherin (CD144), and to further explore its mechanism.</p><p><b>METHODS</b>CD144 in Sup-B15 leukemia cells was stably knock downed via lentivirus-mediated RNA interference (named as Sup-B15/shVEC). The inhibitory effects of IM on Sup-B15/shVEC and Sup-B15 leukemia cells were measured by CCK-8 test, and the apoptosis of those cells was determined by AnnexinV/7-AAD dyeing using flow cytometry, the percentage of CD34⁺CD38⁻ leukemia cells also by flow cytometry. ALDH1 mRNA levels were detected by real-time RT-PCR, and protein levels of CD144, CD133, Bcr-abl and β-catenin by Western blot.</p><p><b>RESULTS</b>IM treatment presented inhibitory effects on Sup-B15/shVEC and Sup-B15 leukemia cells at multiple concentrations of IM. The IC50 of IM on Sup-B15/shVEC and Sup-B15 leukemia cells were 25.1μmol/L and 18.7μmol/L, respectively (P<0.05). After 48h of 20 μmol/L IM treatment, the percentages of apoptosis cell in Sup-B15/shVEC cells and Sup-B15 cell were (13.52±2.06)% and (3.03±0.72) %, respectively (P<0.05). The percentage of CD34⁺CD38⁻ cells in Sup-B15 cells was significantly higher than in Sup-B15/shVEC cells [(2.39±0.28)% vs (0.96±0.07)%, P<0.05). As compared to Sup-B15 cells, the transcription of ALDH1 in Sup-B15/shVEC was remarkably downregulated, and the CD133 protein level was also downregulated in Sup-B15/shVEC cells. Both cytoplasmic and nucleic β-catenin protein levels (but not for Bcr-abl levels) decreased in Sup-B15/shVEC cells as compare to Sup-B15 cells.</p><p><b>CONCLUSION</b>Knockdown of CD144 sensitized Sup-B15 Ph+ ALL cells to IM. The possible mechanisms underlying this phenomenon might be via inhibiting β-catenin nucleic translocation and facilitating β-catenin degradation.</p>