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[ ABSTRACT] AIM: To investigate the protective effect of autophagy on oxidized low density lipoprotein ( ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms .METHODS:The RAW264.7 macropha-ges were pretreated with 3 mmol/L 3-methyladenine (3-MA), 1 μmol/L rapamycin (Rap) or 4 mmol/L 4-phenylbutyric acid ( PBA) respectively for 1 h and then treated with ox-LDL (100 mg/L) for 12 h.The cell viability and apoptosis were determined by MTT assay and flow cytometry with Annexin V-FITC/PI staining, respectively.The activities of lactate de-hydrogenase ( LDH) in the medium and caspase-3 in the cells were determined by detection kits .The protein levels of bec-lin-1 (a molecular marker of autophagy ), glucose-regulated protein 78 (GRP78, an endoplasmic reticulum stress marker) and C/EBP homologous protein ( CHOP, a key-signaling component of endoplasmic reticulum stress-induced apoptosis ) were examined by Western blot .Microtubule-associated protein 1 light chain 3 (LC3, another molecular marker of autoph-agy) was observed under laser scanning confocal microscope .RESULTS: Treatment of the RAW264.7 macrophages with ox-LDL at 100 mg/L for 12 h resulted in significant decrease in cell viability , and dramatic elevation in LDH leakage , cell apoptosis and caspase-3 activity, which were promoted by 3-MA (an autophagy inhibitor) and inhibited by Rap (an autoph-agy inducer ) .ox-LDL induced autophagy in the macrophages as assessed by beclin-1 upregulation and frequent granulation of LC3, which were inhibited by 3-MA and promoted by Rap.Interestingly, 3-MA enhanced, while Rap blocked, the CHOP upregulation induced by ox-LDL.Moreover , PBA ( endoplasmic reticulum stress inhibitor ) significantly inhibited ox-LDL-induced GRP78 upregulation and autophagy as determined by the attenuation of beclin-1 upregulation and frequent granula-tion of LC3.CONCLUSION: Endoplasmic reticulum stress mediates ox-LDL-induced autophagy in macrophages , and moderates activation of autophagy may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression .
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the impact of cryopreservation duration of umbilical cord blood (UCB) on quality of hematopoietic stem cell and outcome of clinical transplantation.</p><p><b>METHODS</b>605 units of UCB which had been used in clinical transplantation were previously cryopreserved for 820 (88-2651) days in average. UCB was detected for total nucleated cell count, CD34+ cells count, cell recovery rate, cell viability and CFU-GM after thawing.</p><p><b>RESULTS</b>No statistical correlation was found between cryopreservation duration and cell recovery rate, cell viability. CFU-GM decreased along with the extension of cryopreservation duration (P=0.011), ranging between 109.6 and 105.7/1 × 10⁵. There was no significant difference on hematopoietic reconstitution time, graft failure, acute GVHD and overall survival among groups with different cryopreservation duration.</p><p><b>CONCLUSION</b>Cryopreservation duration has no significant effect on cell recovery rate, cell viability and clinical transplantation outcome. Extension of cryopreservation duration may reduce CFU-GM of stem cells with fluctaion still in normal range. UCB could maintain cell viability and function to achieve satisfactory clinical transplantation outcome even when thawed after 3 to 7 years' cryopreservation.</p>
Subject(s)
Humans , Cell Count , Cell Survival , Cryopreservation , Fetal Blood , Graft vs Host Disease , Granulocyte-Macrophage Progenitor Cells , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells , Treatment OutcomeABSTRACT
<p><b>OBJECTIVE</b>To evaluate relevant prognostic factors of unrelated single unit umbilical cord blood hematopoietic stem cell transplantation (sUCBT), and to explore the correlation between cryopreservation time of cord blood and cell viability and outcome of sUCBT.</p><p><b>METHODS</b>Retrospective analysis of 137 patients undergoing sUCBT with cord blood provided by Shanghai Cord Blood Bank from Mar. 15, 2007 to Dec. 26, 2013 were performed in this study. The mean cryopreservation time of 137 units cord blood was 698(96-1968) days, with mean cell viability of 87.4% after thawing.</p><p><b>RESULTS</b>No statistical difference on cell viability, hematopoietic reconstitution, graft failure, acute graft versus host disease (GVHD) and overall survival (OS) was found between patients transfused with cord blood preserved below and above 2 years. The 5-year OS of patients transfused with cord blood preserved below and above 2 years were 55.6% and 67.9%, respectively (P=0.124). OS of the UCBT at 2011 and before, and after 2011 was 48.7% and 79.6%, respectively (P=0.001). Age above 16-year-old (RR=2.830, P=0.027) and UCBT at 2011 and before (RR=0.203, P<0.001) were two risk factors of treatment related mortality.</p><p><b>CONCLUSION</b>Outcome of sUCBT in China had significant improvement in recent 2 years. Cryopreservation time of cord blood had no statistical correlation to cell viability after thawing and clinical outcome.</p>