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AIM:To investigate the effects of celecoxib on viability , apoptosis and autophagy in acute myeloid leukemia (AML) cell lines HL-60 and HL-60A.METHODS:The HL-60 cells and HL-60A cells were cultured with vari-ous concentrations (0, 20, 40, 60, 80 and 100μmol/L) of celecoxib.The inhibitory effect of celecoxib on the cell viabil-ity was evaluated by MTT assay .Apoptosis was analyzed by Annexin-V/PI staining.Apoptosis-related and autophagy-relat-ed proteins were determined by Western blot .RESULTS:IC50 of celecoxib were 49.4 μmol/L, 32.0 μmol/L and 25.1μmol/L for HL-60 cells treated with celecoxib for 24 h, 48 h and 72 h, respectively.For HL-60A cells, the corresponding IC50 were 69.1 μmol/L, 42.5 μmol/L and 29.6 μmol/L, respectively.The results of flow cytometry analysis showed the proportions of Annexin-Ⅴ+PI-, Annexin-Ⅴ+PI+and Annexin-Ⅴ-PI+cells were increased in the HL-60 cells, and those of Annexin-Ⅴ+PI-and Annexin-Ⅴ+PI+cells were increased in the HL-60A cells treated with celecoxib for 24 h. After treated with celecoxib , the induction of apoptosis was observed , the apoptosis-related proteins cleaved caspase-3 and cleaved PARP were upregulated , the autophagy-related proteins LC3 II and P62 were both increased , and mTOR, p-mTOR, 4-EBP and p-4-EBP were not changed , indicating that celecoxib inhibited autophagy in the AML cells without the mTOR pathway involvement .CONCLUSION:Celecoxib inhibits the viability of HL-60 cells and HL-60A cells in a time-and dose-dependent manner by its effects of inducing apoptosis and necrosis .Celecoxib inhibits mTOR-independent autoph-agy in AML cells, indicating a possible way of using celecoxib for enhancing the antitumor activity of therapeutic agents to induce cytoprotective autophagy in the AML cells .
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AIM:To construct AFT024-SCF cell line and HPC-Lhx2 cell line for confirming the biological function of AFT024-SCF.METHODS:The HPC-Lhx2 cell line, AFT024-SCF cell line and AFT024-GFP cell line were constructed by retro-viral infection.The expression of stem cell factor(SCF) in AFT024-SCF cells was detected by real-time PCR and Western blot.SCF in the supernatant of AFT024-SCF was detected with ELISA.The supernatant of AFT024-SCF and AFT024-GFP were collected and then diluted (1:10) with basic IMDM medium.So we made 4 culture medium:AFT024-SCF medium was used for experiment group, AFT024-GFP medium was used for endogenous negative control, IM-DM basic medium was used for exogenous negative control, and IMDM basic medium with SCF was used for positive con-trol.SCF-dependent HPC-Lhx2 cell line was cultured in these 4 different medium for 72 h.According to MTT method and colony forming experiment, the biological function of AFT024-SCF was confirmed by the proliferation ability of SCF-depend-ent HPC-Lhx2 cell line.RESULTS:SCF was highly expressed in AFT024-SCF cells.After cultured for 72 h, neither IM-DM basic medium nor GFP-AFT024 medium support HPC-Lhx2 cell line proliferation.However, AFT024-SCF medium supported HPC-Lhx2 cell line expansion as well as the positive control medium.CONCLUSION:AFT024-SCF cells ex-press SCF successfully and recombinant SCF can be replaced by the supernatant of AFT024-SCF culture medium for expan-ding HPC-Lhx2 cell line in vitro.
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Objective To evaluate the clinical efficacy and safety of umbilical cord-derived mesenchymal stem cell (MSC ) treatment of refractory chronic graft-versus-host disease (cGVHD ) after allogene hematopoietic stem cell transplantation. Methods Seven patients developed with cGVHD following allogene hematopoietic stem cell transplantation. Conventional immunosuppressive agent treatment yielded no efficacy. Based upon immunosuppressive agent therapy,umbilical cord-derived MSC treatment was supplemented with a cell density of 1 ×1 06/kg,once a week for consecutive 4 times. Clinical efficacy,safety and survival of the patients were observed. Results Among 7 patients receiving MSC injection,2 obtained complete response (CR)and 3 had partial response (PR)with an overall response rate of 5/7,and the remaining 2 cases achieved no response (NR). No adverse reactions were induced by MSC injection. No patient had primary disease recurrence. One patient developed secondary cytomegalovirus pneumonia after PR and died from severe pneumonia. The remaining patients survived. Conclusions Umbilical cord-derived MSC injection is an efficacious and safe therapy of cGVHD.
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BACKGROUND:Because of their immunological properties, bone marrow mesenchymal stem cells transfusion is developed as a new treatment for refractory graft-versus-host disease. OBJECTIVE:To analyze the safety and curative effect of bone marrow mesenchymal stem cells transfusion on treating different organ damages in graft-versus-host disease after al ogeneic hematopoietic stem celltransplantation. METHODS:Eight patients with malignant hematologic disease were included in this study. The patients developed severe steroid-resistant graft-versus-host disease after al ogeneic hematopoietic stem celltransplantation and received transfusion of mesenchymal stme cell(1×106 of immunosuppressive agent. RESULTS AND CONCLUSION:For the total y eight patients, six got response (two cases of complete remission, and four cases of partial remission) and two showed no remission. Four of five cutaneous damages were ameliorated and one showed no effect. For three cases of oral graft-versus-host disease, two acquired complete remission and one showed partial remission. Two cases of liver graft-versus-host disease and two cases of astro-intestinal graft-versus-host disease obtained complete remission. No response was displayed to three cases of ocular graft-versus-host disease, one case of bronchiolitis obliterans, and one case of urinary graft-versus-host disease. In the median fol ow-up of 28 months (7-62 months), three patients developed posttransplant lymphoproliferative disorders within 3 months after mesenchymal stem cells transfusion. Administration of mesenchymal stem cells is safe for treatment of severe graft-versus-host disease after al ogeneic hematopoietic stem celltransplantation. Mesenchymal stem cells transfusion may be a promising/kg) together with the primary therapy therapy for refractory cutaneous , astro-intestinal, liver and oral graft-versus-host disease but not for pulmonary, ocular and urinary graft-versus-host disease. Whether mesenchymal stem cells transfusion is associated with posttransplant lymphoproliferative disorders needs more case data.
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Objective To investigate the influences of cytarabine on Survivin gene expression in human leukemia K562 cell line and discuss the mechanism of drug resistance in chemotherapy. Methods The IC50 of cytarabine was chosen by MTr assay. The K562 cells were exposed to certain concentration of cytarabine for about 24 hours and 48 hours. The expression levels of Survivin gene were detected by RT-PCR and Western-blot. Results After exposed to cytarabine for 24 hours and 48 hours, the Survivin mRNA level of K562 cells was significantly elevated about 1.92 and 3.38-fold, and the protein expression level was elevated about 1.92 and 2.64-fold. Conclusion An elevated expression of Survivin was tested in K562 cells treated by cytarabine. Consequently, the elevated expression of Survivin could resist apoptosis induced by chemotherapeutic agent which probably associated with chemotherapeutic drug resistance.
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Objective To investigate the influence of the PBSC collection yield by choosing the differ-era venous accesses in the healthy donors. Methods 118 healthy PBSC donors performing PBSC collection between January 2000 and December 2007 in our hospital were divided into four groups according to the differ-ent venous accesses. The PBSC collection yield of four groups,including mononuclear cells (MNC) count and CD34+ cells count were observed. Results In the ulnar V-ulnar V group,MNC (5.31±2.29)×108/kg,CD34+ cells (4.78±2.06)×106/kg;ulnar V- antecubital V group,MNC(5.11±2.34)×108/kg,CD34+cells(4.34±1.99)×106/kg;antecubital V- antecubital V group,MNC (5.61±1.73)±×108/kg,CD34+cells (4.60±1.42)×106/kg;ulnar V- radial V group,MNC(4.60±×1.70)×108/kg,CD34+cells (4.05±1.50)×106/kg.There was no statistical differ-ence of the PBSC collection yield between four groups (P>0.05). Conclusions Different venous accesses don't affect the PBSC collection yield in the PBSC healthy donors.
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AIM: To induce rat bone marrow mesenchymal stem cells (BMSC) into cardiomyocytes and investigate the influence of serum coming from acute myocardial infarction (AMI) rat on the procedure. METHODS: The passage 3 BMSC were divided into six groups: groupⅠwas control group; groupⅡwas induced with 5-azacytidine; group Ⅲ was induced with 5-azacytidine and serum from AMI rat; group Ⅳ was induced with 5-azacytidine and serum from normal rat; group V and group Ⅵ were induced with serum from AMI rat or normal rat. The cardiac troponin T, GATA-4 and desmin were detected 30 days after induction. RESULTS: After inducing by 5-azacytidine, 5-azacytidine and two kinds of serum, some cells in the three groups differentiated into cardiac like cells. The expressions of cardiac troponin T, GATA-4 and desmin were positive in cells differentiated from BMSC. The troponin T expression in control group and group inducing by AMI serum alone were negative but GATA-4 and desmin expressed weakly. Some cells induced with 5-azacytidine and serum were slowly beating 2 weeks after induction, but the cells induced with 5-azacytidine alone was not beating.CONCLUSION: Serum from AMI can not induce BMSC to differentiate into cardiomyocytes, but it promotes BMSC differentiate into cardiomyocytes induced by 5-azacytidine and facilitate the differentiated cells to mature.
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AIM: To re-identify the special motif regulating osteoclast(OC)differentiation in receptor activator of nuclear factor kappa B(RANK)to provide evidences for studying the mechanism of OC differentiation.METHODS: Eight amino acids were mutated(from DIIVVYVS into ELLAAFAA)in the fragment between the 533th and the 540th amino acids in RANK cytoplasmic domain.Eight mutant TNFR1/RANK chimeras,each consists of TNFR1(tumor necrosis factor receptor 1)extracellular domain linked to transmembrane domain and cytoplasmic domain of RANK with one amino acid mutated in cytoplasmic domain was constructed by point mutation method.After the eight mutant chimeras were finished,they were packed with plat E cell line to produce the retrovirus expressing mutant TNFR1/RANK.The bone marrow macrophages(BMMs),isolated from TNFR1/R2 double knockout mice,were infected with retrovirus derived from different mutants and infected BMMs which did not differentiated into OCs were inspected after stimulated by TNF-? and M-CSF.The fragment consisted of different amino acids in TNFR1/RANK chimeras,which couldn't induce OC formation after mutated,may be the special motif regulating OC differentiation.RESULTS: We found that all BMMs transfected by TNFR1/RANK-533,TNFR1/RANK-539 or TNFR1/RANK-540 differentiated into OCs,indicating that none of amino acids D533,V539 or S540 had an effect on OC differentiation.A fewer of BMMs transfected by TNFR1/RANK-534 differentiated into OCs,indicating that I534 had a partial effect on OC formation.Most importantly,BMMs transfected TNFR1/RANK-535,TNFR1/RANK-536,TNFR1/RANK-537 or TNFR1/RANK-538 did not differentiated into OCs,indicating each of amino acids I535,V536,V537 and Y538 played a pivotal role in OC differentiation.CONCLUSION: The amino acid fragment consists of I534,I535,V536,V537 and Y538 may be the special motif regulating OC differentiation in RANK.