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Objective This study aims to investigate the impact of cultivation time on dendritic cells(DCs)and their derived exosomes′ expression of immune-related membrane proteins(CD80,MHC-Ⅰ,MHC-Ⅱ)and provides experimental evidence for future research.Methods Mouse bone marrow cells were induced to differentiate into DCs using GM-CSF and IL-4,followed by maturation stimulation withTNF-α.Exosomes were extracted using ultracentrifugation.Western blot and Amnis image flow cytometry were used to identify exosomes derived from mouse DCs.Amnis image flow cytometry was used to detect the expression of immune-related proteins CD80,CD11c,MHC-Ⅰ,and MHC-Ⅱ in mouse DCs and their exosomes.Results After 5 days of in vitro cultivation,more than 50%of dendritic cells expressed CD80,CD11c,MHC-Ⅰ,and MHC-Ⅱ,reaching the highest level on day 13.The positivity rates were as follows:CD80(97.29±0.63)%,CD11c(92.31±1.18)%,MHC-Ⅰ(97.91±0.49)%,and MHC-Ⅱ(97.91±0.49)%.The differences were statistically significant(P<0.001).The expression gradually decreased after day 13,but approximately 80%of DC cells still expressed MHC-Ⅰ and MHC-Ⅱ immune molecules on day 30.The expression levels of CD80,CD11c,and MHC-Ⅱ on the exosome membrane were highest on day 5 and then decreased overall with prolonged cultivation time,except for MHC-Ⅰ molecules.The differences were statistically significant(P<0.01).Conclusions In vitro-cultured mouse dendritic cells express high levels of immune-related membrane proteins and can be stably maintained for a long time under suitable culture conditions.The secreted exosomes also carry abundant immune-related membrane proteins,but no significant correlation was found between the immune-related proteins on the dendritic cell surface and the exosome membrane surface.
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Objective:To investigate the role of stress-inducible protein Sestrin2 (Sesn2) in the improvement of insulin resistance in rat L6 skeletal muscle cells treated with liraglutide.Methods:The establishment of insulin resistance model of rat L6 skeletal muscle cells was induced by palmitate. The experimental cells were divided into control group(Con group), palmitate 0.6 mmol/L treatment group(PA group), palmitate 0.6 mmol/L+ liraglutide 10 nmol/L treatment group(PA+ Lir10 group), palmitate 0.6 mmol/L+ liraglutide 100 nmol/L treatment group(PA+ Lir100 group), and palmitate 0.6 mmol/L+ liraglutide 1 000 nmol/L treatment group(PA+ Lir1000 group). The cell counting kit 8(CCK8) method was used to detect the cell activity in each group. Western blotting was used to detect the expression levels of glucose transporter 4(GLUT4), protein kinase B(Akt), phosphorylated protein kinase B(p-Akt), and Sesn2 protein in L6 cells. L6 cells were transfected with siRNA to inhibit the expression of Sesn2. The cells were treated with palmitate and liraglutide. Western blotting was used to detect the expression levels of Sesn2, Akt, p-Akt, and GLUT4 protein in L6 cells.Results:Compared with Con group, the cell survival rate, p-Akt/Akt ratio, Sesn2, and GLUT4 protein expression in PA group decreased significantly( P<0.05). After liraglutide intervention, the cell activity, p-Akt/Akt ratio, Sesn2, and GLUT4 protein expression of PA+ Lir100 and PA+ Lir1000 groups was increased( P<0.05). After inhibiting the expression of Sesn2, p-Akt/Akt ratio and GLUT4 protein in transfected si-Sesn2 and treated with 0.6 mmol/L palmitate group(PA+ si-Sesn2 group) and transfected si-Sesn2 and treated with 0.6 mmol/L palmitate+ liraglutide 100 nmol/L group (Lir100+ PA+ si-Sesn2 group) were significantly lower than those in transfection negative group (si-Con group; P<0.05). Even after liraglutide intervention, compared with PA+ si-Sesn2 group, p-Akt/Akt ratio and GLUT4 protein expression level were not significantly increased in Lir100+ PA+ si-Sesn2 group ( P>0.05). Conclusions:Palmitate could induce the decrease of p-Akt/Akt ratio and GLUT4 protein expression in L6 cells. Liraglutide upregulates the expression of Sesn2, which leads to the increase of p-Akt/Akt ratio and GLUT4 protein expression and contributes to the improvement of insulin resistance.
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Objective:To observe chicken typeⅡcollagen-induced arthritis( CIA) in serum of mice with the dynamic changes of IL-27,IL-17,IL-10. Methods: 54 DBA1/J mice were randomly divided into control group(n=6) and model group(n=12), according to the progress of CIA mouse course early,middle and late(7,14,and 35 days after booster immunization) ,taking the eyeball for blood and separating the serum under sterile condition. The dynamic levels of cytokines IL-27,IL-17,IL-10 were detected by flow cy-tometry. Results:The level of IL-27 in the model group was significantly declined in the the progress of CIA mouse course early and middle(P 0. 05);the level of IL-17 in the disease early and late was no significant difference(P> 0. 05) compared with the control group,while in the mid course significantly higher than control group(P 0. 05). Conclusion: IL-27,IL-17,IL-10 paticipate in the pathogenesis of CIA and their alterations at different stages of the disease have a relation to the development of arthritis.
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BACKGROUND:To reduce the amount of bleeding and the amount of hemoglobin after total knee replacement has been a key project in the clinical research in the division of bone and joint. Currently, ice therapy has been widely used in the clinic for tissue sweling and pain due to various physical and chemical factors. OBJECTIVE:To investigate the risk factors of postoperative hemoglobin after total knee replacement and discuss the effects of ice intervention. METHODS: 240 patients with osteoarthritis based on the random draw principles were equaly divided into the treatment group and the control group. The general information, disease status, diagnosis and treatment and prognosis of the two groups were investigated. Al patients were actively subjected to artificial total knee replacement. On the basis of the treatment in the control group, the treatment group received ice intervention at 2 hours after replacement for 7 consecutive days. RESULTS AND CONCLUSION:The postoperative hemoglobin decrease occurred in 34 patients, with the incidence of 14.2% among 240 patients at 7 days after replacement. Multivariate logistic regression analysis results showed that age, no ice treatment, body mass index were the main risk factors for hemoglobin decrease after total knee replacement (P 0.05). Results verify that clinical application of total knee replacement facilitated the knee recovery in patients with osteoarthritis, but hemoglobin decrease and bleeding existed. Active ice intervention can reduce the risk and relieve postoperative pain.