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Objective To examine whether Tim-3 plays a protective role in paraquat poisoning induced excessive immune response and tissue damage based on the critical roles of Tim-3 controlling inflammatory response.Methods A paraquat poisoning model was established in wild type and in Tim-3 transgenic C57BL/6 mice by intraperitoneal injection of paraquat (40 mg/kg) .In addition, C57BL/6 mice with paraquat poisoning were injected with Tim-3 soluble protein( sTim-3) or control protein to see the effect of Tim-3 blocking on the progression of paraquat poisoning.Samples were collected at 6 and 24 h after paraquat injection respectively and were examined for tissue damage, cytokine expression and paraquat metabolism.Results After paraquat poisoning, there was significantly attenuated tissue damage in the lungs and kidneys and decreased TNF-α,IL-6 and IL-1 beta expression in the PBMCs or in the serum from Tim-3 transgenic mice compared to wild type mice.The serum concentration of paraquat in Tim-3 transgenic mice was also significantly decreased.However, in sTim-3 treated paraquat poisoning mice, there was significantly increased cytokine expression and tissue damage compared to control protein treated mice.The in vitro data showed that Tim-3 signaling negatively regulated macrophages mediated inflammatory response.Conclusion Tim-3 plays a critical role in maintaining the homeostasis after paraquat poisoning. Further investigation on the regulatory roles of Tim-3 in inflammation will shed new light on the pathogenesis of paraquat poisoning and provide new therapeutic strategies.
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Objective To develop a human Tim-3 specific monoclonal antibody and evaluate its biological activity and possible use in clinical diseases associated with dysregulated Tim-3 expression .Methods The BALB/c mice were immu-nized by conventional method, and positive clones were used to develop anti-human Tim-3 antibody, the binding and neutralization activities of which in vitro and in vivo were investigated.Results ①A monoclonal antibody (clone L3D) which could specifically bind to human Tim-3 protein in ELISA assay was obtained and the subtype of the monoclonal antibody was IgG2a .②Flow cytometry indicated that the monoclonal antibody could bind to Tim-3 expressed in human U937 cells.This antibody also showed a cross activity to mice′Tim-3.③The monoclonal antibody inhibited the apoptosis of THP1 cells induced by Gal-9, the ligand of Tim-3.④Injection of Tim-3 antibody exacerbated sepsis in mice as marked by the decreased survival rate and increased expression of pro-inflammatory cytokines .Conclusion An anti-human Tim-3 monoclonal antibody is successfully obtained.The excellent binding and neutralization activities of this antibody enable it to be widely used in clinical diseases associated with deregulated Tim-3 expression .
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Objective:To study the effects of the Gekko gecko ethanol extract on granular cells apoptosis of rat ovaries.Methods :3 month and 6 month female Wistar rats were taken in this study, and 20 rats were in each age group.Each age group were randomly divided into two groups(experimental group and control tragastric administration in experimental group, corresponding volume of 0.9% sodium chloride was given in control group by the same way.Apoptosis of granular cells and distribution of cell cycle in rat ovary were measured by terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling (TUNEL) and propidi-um iodide(PI) staining, AnnexinV/PI double labelling staining by flow cytometry (FCM) after administration for 15 days.Re8utts:The apoptosis numbers of granular cells in 3 month and 6 month age experimental group were 34 ±7 and 53 ±11, in control group were 48±9 and 76±17 respectively by TUNEL.There was signrficantly statistic difference (P <0.01).The apoptosis rates in 3 month and 6 month age experimental group were(6.06 ±2.01)% and (12.16 ±3.26) % , in the control group were (8.23±2.13) % and (23.69 ±6.28)% respectively by PI staining.There was significantly statistic drfference (P <0.05).However, no significant difference was found between two groups in cellular cycle distribution (P > 0.05).Early apoptosis rates in 3 month and 6 month age experimental group were (6.71±3.11) % and (23.74±6.28) % , in the control group were (19.05 ±5.78) % and (36.55± 8.35) % respectively by AnnexinV/PI double labelling staining FCM.There was significantly statistic difference (P <0.01).The apoptosis of granular cells were sig-nificantly less in experimental groups comparing wrth control groups both in 3 month and 6 month age group (P<0.01 or P<0.05).Conclusions :The ethanol extract of gekko gecko can inhibit the apoptosis of granu-lar cells in rat ovary, then improves the ovary function and delay ageing of rat.
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