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Objective To investigate the effect of hypoxic preconditioning on anti-inflammatory responses of bone marrow mesenchymal stem cells (BMSCs) in rats through in vitro and in vivo experiments.Methods In vitro experiment The isolated rat BMSCs were cultured by whole bone marrow adherence method.The cells at passage 3 were seeded in 24-well plates at a density of 1 × 106 cells/ml and randomly divided into 5 groups (n =8 wells each) using a random number table:control group (group C),normoxia-incubated group (group N),hypoxic preconditioning group (group H),hypoxia preconditioning + STAT3 inhibitor Stattic group (group HS) and hypoxia preconditioning + anti-IL-10 monoclonal antibody group (group HA).In group C,BMSCs were incubated in DMEM culture medium.In group N,BMSCs were exposed to21％ O2-74％ N2-5.0％ CO2 for48 h.In group H,BMSCs were exposed to 0.5％ O2-94.5％ N2-5.0％ CO2 for 24 h followed by 24 h exposure to normoxia.In HS and HA groups,500 μg/ml Stattic and 100 μg/rnl anti-IL-10 monoclonal antibody were added to the culture medium before hypoxia preconditioning,respectively.The expression of phosphorylated STAT3 (p-STAT3) and IL-10 was determined by Western blot.In vivo experiment Healthy male Sprague-Dawley rats,weighing 300-350 g,in which intrathecal catheters were successfully implanted without complications,underwent spinal cord ischemia by occlusion of the thoracic aorta combined with controlled hypotension.Three hundred rats with spinal cord I/R injury were randomly divided into C,N,H,HS and HA groups (n =60 each) using a random number table.Immediately after onset of reperfusion,DMEM medium 300 μl was injected intrathecally in group C,and BMSC suspension 300 μl (1 × 106 cells/ml) was injected intrathecally in N,H,HS and HA groups.Neurological function was scored before ischemia and at 4,12,24 and 48 h of reperfusion (T0-,).The animals were then sacrificed and the lumbar segment of spinal cord was removed for detection of the content of IL-10,TNF-α,IL-1β,IL-6,monocyte chemotactic protein-1 (MCP-1),and macrophage inflammatory protein-1 α (MIP-1 α) (by ELISA) and the number of activated microglia (by immuno-histochemistry).Results Compared with C and N groups,the expression of pSTATβ and IL-10 was significantly up-regulated,the neurological function score and IL-10 content were increased,the content of TNF-α,IL-1β,IL-6,MCP-1 and MIP-1α and the number of activated microglia were decreased in group H (P ＜ 0.05).Compared with group H,the expression of p-STAT3 and IL-10 in group HS and expression of IL-10 in group HA was significantly down-regulated,and the neurological function score and IL-10 content were decreased,and the content of TNF-α,IL-13,IL-6,MCP-1 and MIP-1α and the number of activated microglia were increased in HS and HA groups (P ＜ 0.05).Conclusion Hypoxic preconditioning can enhance anti-inflammatory effects of BMSCs,thus increasing BMSCs-induced reduction of spinal cord ischemia-reperfusion injury in rats.
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Objective To establish a off-pump leukocyte depletion method in canines.Methods Twenty-one healthy adult mongrel dogs of both sexes, weighing 10-12 kg, were randomized into 2 groups: control group (group C, n = 9) and leukocyte depletion group (group LD, n = 12) . An extracorporeal leukocyte filtration end was constructed by aseptic puncture of the bilateral external jugular veins via a blood transfusion line and the leukocyte filter for extracorporeal leukocyte depletion. In group LD, blood was filtered with a MP-300 blood line and a SQ40S leucocyte depletion filter one end placed in the right external jugular vein (artery end) and the other end placed in the left external jugular vein (venous end). After heparin anticoagulation, a MP-300 blood line pump was used as the power. Blood was filtered at a rate of 75 ml/min and it was maintained for 60 min. The artery end was then closed, normal saline injected into the closed circuit, the remaining blood pumped into the body, and then the venous end closed. In group C, aseptic puncture of the bilateral external jugular veins was performed. Arterial blood samples were taken immediately before leukodepletion (baseline, T_0 ) , at 10, 20, 30, 40, 50 and 60 min of leukodepletion (T_(1-6) , depletion period), and at 10, 20, 30, 40, 50, 60, 120, 180 and 270 min after leukodepletion (T_(7-15) , recovery period) for determination of blood routine. MAP, HR, RBC.Plt and body temperature were recorded at T_0 , T_6 , T_(12) and T_(15) . Results There were no significant difference in MAP and RBC between the two groups ( P > 0.05 ) . HR, body temperature, and Plt were significantly lower in group LD than in group C (P < 0.05) .The leukocyte concentration was lower during depletion period in group LD than in group C ( P < 0.05) , while there was no significant difference in leukocyte concentration during recovery period between the two groups (P > 0.05 ). Conclusion The off-pump leukocyte depletion method is successfully established and has exact efficacy with less adverse effects in this study.
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To study the effects of salidroside-pretreatment on changes of neuroethology in rats with global cerebral ischemia-reperfusion injury so as to investigate its probable mechanism.
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Objective To evaluate of red blood cell as giving instruction in whole white blood cell immunological activity by new nature experimental system of hemaimmune reaction rood map. Methods Plasma 0.3ml were added to whole blood cells (including: red blood cell and white blood cells) or white blood cells 0.2ml, and incubated for 1h at 37℃. The content of IL-8 and IL-12 was determined by enzyme linked immunadsorbent assay (ELISA) method. The expression level of CD4, CD8, CD35 and CXCR4 on white blood cells was determined by method of Flow Cytometry. Results The content of IL-8 (5.96?4.26) and IL-12 (9.84?2.23) in whole blood and plasma nature group was significantly lower than that (13.59?3.69?B?pg~ -1 ?ml~ -1 ) and (15.09?9.86?B?pg~ -1 ?ml~ -1 ) in white blood cell and plasma isolation group (P
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Objective To study the changes in the expressions of CXCR4 of leucocytes as modulated by erythrocytes from cord blood. Methods 0.2ml suspension of whole blood cells (or 0.2ml suspension of leucocytes) was added to 0.3ml of auto plasma, and 0.2ml suspension of S180 (5?10~6/ml) was added as stimuli. The suspension was incubated at 37 ℃ for 1 hour. Normal saline instead of S180 was used as control The expression changes in CD35 on erythrocytes and CXCR4 on leucocytes were determined with flow cytometry assay. The data could be grouped under four heads: (1) cancer cells 0.2ml were added to whole blood cells 0.2ml and plasma 0.3ml; (2) NS 0.2ml were added to whole blood cells 0.2ml and plasma 0.3ml; (3) cancer cells 0.2ml were added to white blood cells 0.2ml and plasma 0.3ml; (4) NS 0.2ml were added to white blood cells and plasma 0.3ml. Results When activated by S180, the expression of CD35 on cord erythrocytes was decline not so low as that of adult′; while the expression of CXCR4 on cord leucocytes was increased not so high as that of adults′. When activated by S180, the expression of CD35 on adult erythrocytes was significantly decreased than that of group of not activating (40.21%?11.52% and 28.65%?8.08% respectively), while the expression of CXCR4 on adult erythrocytes was significantly increased (40.62%?17.89% and 64.18%?11.69% respectively, P