The Mechanism Research Of Thrombocytopenia Induced By LPS On Mouse

Object:Thrombocytopenia(TCP) is one of sever complications of sepsis,which always increa- ses the bleeding tendency of patients.Meanwhile it is a evaluation index of the severity degree of sepsis.But,the mechanism of TCP accompanied with sepsis is unclear.Existing research results show that the occurence of TCP during sepsis is multifactor,which may be involved in inflammatory response,coagulation system and immune response.Assaulting mice with lipopolysaccharide(LPS) from Escherichia coli can induce rapid TCP.While it is ambiguous that the occurence of TCP is the result of either inflammatory response induced by LPS or overactivation of coagulation system,nor the direct destroy on platelet after interactivation between LPS and Toll-like receptor 4 on platelet.Therefore,through establishing the mouse TCP model by injecting different dose of LPS intraperitoneally,we observe the variation tendency of the rapid TCP induced by LPS,assay the level of activated markers of inflammatory response and coagulation system and the influence on apoptosis indexes from LPS on isolated platelets,estimate the relationship between TCP and the state of inflammatory response and coagulation system existed in TCP induced by LPS,and the effects of the survival state of platelet changed by the direct interaction between LPS and platelet. Content1. Establishment and appraisement of mouse TCP model induced by LPS injection once intraperitoneally.2. The research about the state of inflammatory response and coagulation system of mouse TCP induced by LPS and the relationship of them with TCP.3. The research of platelet apoptosis induced by LPS in vitro. MethodPart Ⅰ:Establishing mouse TCP model by injecting LPS once intraperitoneally, observing the change of mouse PLT,MPV and PDW at different time after injecting different does of LPS intraperitoneally.Part Ⅱ:4h and 24h after injecting non-lethal dose(0.5mg/kg) and lethal dose ofLPS intraperitoneally,collecting samples,assaying the concentration of IL-6,TNF-a, TAT,FDP,D-Dimer and TPO in plasma using ELISA,observing the histopathologic morphology change in lung,and testing the expression level of TPO mRNA in kidney.Part III:Preparing washed platelet,co-incubating wtih LPS under room tempe-rature for 30min,then assaying ATP level and Caspase-3 activity using chemi-luminescence,testing the PS exposure and △ψ depolarization using flow cyto-metry,detecting the expressions of Caspase-8、-9、 Bak、Bax and Bcl-2 using Western Blot. ResultsPart I:Injecting LPS once intraperitoneally can induce mouse TCP steadily,and the degree of TCP depends on the dose of LPS.According to the effect on mortality the dose of LPS is devided into non-lethal dose(0.05mg/kg-5mg/kg) and lethal dose(50mg/kg).The differences of PLT between different groups of non-lethal dose have statistical significance,nor of MPV and PDW.While,analysizing all groups in 24h,include lethal dose group,the differences with statistical significance were founded between groups interms of MPV and PDW.Part II:Compared with control group,the concentrations of IL-6 and TNF-a in different groups dealed with different dose of LPS decreased apparently,but there’s no difference wtih statistical significance was founded between different LPS groups,which indicated that the mouse body was not in a state of overinflammatory response at designated time, and there was no difference was founded in terms of the inflammatory response state between different LPS group at the same time.Compared with control group, TAT level of LPS groups decreased apparently,while no difference was founded between LPS groups,which indicated that there is no difference in terms of the active state of thrombin between LPS groups,although LPS can result in sharp decrease of TAT. Compared with control group,the D-Dimer and FDP concentration of LPS groups was no apparent decrease,and no difference was founded between different dose LPS groups.This indicated that there was no overactivity and hyperfibrinolysis in mouse coagulation system.There was no difference between group in terms of plasm TPO concentration and expression of TPO mRNA inkidney,which showed that the mouse body did not compensate through upregulating the production of TPO.Part Ⅲ:Compared with control group which act as negtive control and wtih thrombin group which act as positive control,after co-incubation with LPS,the increasement of ATP,PS expose,△ψ depolarization,the increasement of Caspase-3 activity and the upregulation of Caspase-8,-9,Bak and Bax were detected. ConclusionPart Ⅰ:Injecting LPS once intraperitoneally,can induced stable mouse TCP model.The degree and duration of TCP depends on the dose of LPS.Meanwhile,the body can initiate rapid compensatory mechamism to increase the production of PLT.Part Ⅱ:TCP induced with single injection using LPS dose not accompanied with overactive inflammatory response and coagulation system,this showed us that LPS can induce TCP through a pathway independent on overactive inflammatory response and coagulation system.The rapid compensatory relaese response of PLT was mediated through TPO-independent pathway.Part Ⅲ:In vitro,LPS can induce platelet apoptosis characterized with △ψ depolarization,the increasement of Caspase-3 activity and the upregulation of Caspase-8,-9,Bak and Bax expression.This apoptotic transform may occur secondary to the over activation of PLT by LPS.