The Expression And Treatment Of TREM-1 In Mice Of Severe Acute Pancreatitis

Severe acute pancreatitis is a common emergency in clinic with complex etiology and pathogenesis and is difficult to treat.The mortality rate is still as high as 20%-30%,and the exploration on its treatment has always been a hotspot.C-reactive protein and procalcitonin has already been used to predict infection and prognosis in clinic, but the value is very limited. One of the main problems is that there is no specific indicator in serum which can accurately diagnose and prognosticate SAP. A sensitive and specific indicator is required to be found in monitoring disease progression and guiding the treatment.The triggering receptor expressed on myeloid cells (TREM) is a member of the immunoglobulin superfamily.The understanding of the process in inflammatory response,including its start and development, is further understood since the first discovery of TREM-1 in 2001. TREM-1 is mainly distributed in the peripheral blood neutrophil leukocyte and monocytes subsets,which are natural immune response cells.The expression of TREM-1,identifying specifically the surface receptor of pathogens, is also selectively expressed in the phagocytes,the pulmonary, intestinal fluid and other body fluids.TREM-1 is expressed in many inflammatory diseases including infectious,non-infectious inflammation and cancer, etc. TREM-1 can promote the secretion of pro-inflammatory cytokines in mice, induced by neutrophils and monocytes secrete neutrophil chemotactic factors,such as IL-8,monocyte chemoattractant protein (MCP-1,MCP-3) and macrophage inflammatory protein-1α(MIP-1α).TREM-1 activation can lead to rapid neutrophil degranulation,respiratory burst and phagocytosis.Toll-like receptors(TLR)-2 orTLR-4 and ligand recognition may be activated after TREM-1 promote the release of a large number of proinflammatory cytokines, while inhibiting the release of IL-10.TREM-1 with these downstream cytokines form a positive feedback autocrine loop regulation to enhance the inflammatory response. TREM-1 mediated signal transduction pathway in the inflammatory response cascade and the incidence of sepsis plays a pivotal role. Gibot constructed, respectively, the Wistar rat model and the mice model of sepsis.They synthesized the TREM-1 extracellular parts of mice and rats,and the synthetic peptide of murine extracellular domain could protect mice from death of endotoxin,while in the rat model,the use of synthetic peptides in rat extracellular can improve the hemodynamic status to reduce the development of lactic acidosis and regulate the release of pro-inflammatory cytokines,such as TNF-α, IL-1β.It could improve survival. The results showed that the extracellular domain peptides may be an effective target for the treatment of sepsis.This research plans to analyze the TREM-1 gene expression on mRNA level in mice model of SAP and to detect protein expression with immunohistochemistry to assess its role in the course of SAP.It plans to conduct the TREM-1-IgG fusion protein with prokaryotic expression vector, expressed in E. coli and purified the fusion protein for the treatment of SAP in mice, which demonstrate that the TREM-1 may be a molecular therapeutic targets in the treatment of SAP.It was valuable for obtaining a better understanding of TREM-1 in the pathway of signal transduction and TREM-1 as a diagnostic marker,may be valuable for various diseases and It may be helpful in exploring new treatment of related diseases.1.The expression of TREM-1 in mice with SAPObjective:To detect the expression of TREM-1 in mice with severe acute pancreatitisMethods:Male kunming mice(n=50) were randomly divided into 5 groups. Kunming mice were injected intraperitoneally with 20% L-arginine in two doses of 4 g/kg each,1 h apart. Mice in control group received intraperitoneally injections of same amount of saline.Mice were sacrificed at 24h,48h,72h and 96h after SAP induction. Serum amylase, creatinine, and ALT were examined at varying time points after injection and pathological evaluation of pancreatic tissues was performed. The expression of TREM-1 mRNA in pancreatic tissues and peripheral blood leucocyte were determined by RT-PCR,real-time PCR. The expression of TREM-1 protein was identified by immunohistochemistry.Results:Detection of serum amylase and histopathology confirmed the successful modeling. Mice in each group made the point in time in accordance with the disposal of specimens from pancreatic tissue under aseptic technique, extracted total mRNA, reverse transcription-polymerase chain reaction (RT-PCR) amplification of a mouse TREM-1, amplified product gene in line with expectations, recovery purified product submission sequenced, sequencing results is the same as GenBank published sequences. The real-time PCR results showed that TREM-1 expression with the progression of the pancreas in various tissues of mice have also changed.This research confirmed that the TREM-1 protein was expressed in pancreatic tissues with immunohistochemistry.Conclusions:It was observed that the TREM-1 content increased at the first 24 h and a continued increase up to 48h.TREM-1 returned to normal level after 48h. In the occurrence and development of severe acute pancreatitis, TREM-1 plays an important role,and varies with the course of thedisease.2.Murine TREM-1 extracellular domain and human IgG Fc fragment fusion protein:recombinant expression and purification and identificationObjective:To construct mouse TREM-1-IgG fusion protein prokaryotic expression vector, expression and purification and identification of recombinant mouse TREM-1-IgG fusion protein.Methods:The use of restriction endonuclease sites EcoR I and XhoⅠ, double-digested prokaryotic expression vector pMD-18T.The pMD 18-T/mouse TREM-1 extracellular and pMD-18T/human IgG-Fc plasmid were respectively cut down by the EcoR I/BamH I and BamH I/Xho I; The three fragments after digestion were linked by the T4DNA ligase,and then were transformed into E.coli DH5a. After the extraction of recombinant plasmid pGEX4T-1/TREM1-IgG, we identified the positive clones.The vector of pet-28a-His-sumo was selected as the expressive vector.The target fragment was amplified through RT-PCR and was cut down by BglⅡ/XhoⅠ.The fragment was inserted into the pet-28a-His-sumo in order to construct the prokaryotic expressive vector pet28a/murine TREM-IgG. Positive clone which was identified by DNA sequencing. was transformed into BL21.The protein was induced by IPTG through the optimization of expression conditions,His-affinity-resin chromatography was used to purify the protein.The preliminary purified fusion protein was murine TREM-1-IgG concluding the carrier protein,SUMO.The protein concluding SUMO can be specifically removed SUMO by the specific SUMO protease.Purified protein was identified to be the TREM-1 protein with SDS-PAGE and Western-Blot analysis.Results:The recombitant plasmid pGEX4T-1/murineTREMl-IgG was transformed intoE.coli DH5a.The plasmid was extracted and cut down by the specific enzymes.The fragments were identified through the DNA sequencing。The expression product was confirmed to be TREM-1 fusion protein by SDS-PAG and Western Blot.We replaced pGEX4T-1 with pet-28a.It was transformed into BL21 and induced by IPTG,and then a large number of tprotein was expressed The protein concluding SUMO can be specifically removed SUMO by the specific SUMO protease.The purified expression product was also identified by SDS-PAG and Western Blot.Conclusions:The murine TREM-1-IgG fusion protein expression vector was successfully constructed. The purified protein was identified by SDS-PAGE and the Western-Blot. 3. The efficacy study of the TREM-1 fusion protein in the treatment of SAP miceObjective:To observe the effect of the TREM-1 fusion protein in the treatment of SAP miceMethods:70 Kunming mice,each by intraperitoneal injection of L-arginine to build severe acute pancreatitis model,were randomly divided into seven groups.The first group and the second group were of TREM-1 treatment groups which 6 hours after modeling via the tail vein injection of TREM-1 recombinant fusion protein, administered concentration 8mg/kg, the third and forth groups for the positive control group,and an other for negative control group using Ulinastatin which is used in clinical, administered a concentration of 100,000U/kg, fifth and sixth groups were of the SAP control group, normal saline intravenously.The first, third, fifth groups respectively were sacrificed in 24 hours after modeling, second, forth and sixth respectively, were killed 72 hours after modeling.Detection of the expression of inflammatory cytokines in peripheral blood and pathological analysis of the pancreas and lung damage, assess the value of TREM-1 recombinant fusion protein.At the same time immunohistochemical was used to observe the therapy effect.Results:The overall mortality rate of TREM-1 and Ulinastatin treatment group were significantly lower than SAP groups (p<0.05);inflammatory factors (MIP-la,sTREM-1 and ultra-sensitivity CRP) were significantly lower than SAP group (p<0.05)。Pathological analysis showed that the damage of pancreas and lung were allieviated in TREM-1 groups(p<0.05) than SAP control group; Immunohistochemistry of NF-κB in pancreas and lung showed that the protein expression levels were decreased in TREM-1 group(p<0.05) and the efficacy was nealy equal to Ulinastatin.Conclusion:TREM-1 fusion protein effectively alleviated the inflammatory response in mice with SAP.It was observed that the damages in pancreas and lung were alleviated It also decreased the inflammatory factor content in serum.This study came to the following conclusions:1. TREM-1 involved in the occurrence and development of severe acute pancreatitis in the process of inflammation,TREM-1 in the pathogenesis of SAP mice 24h, a continued increase up to 48 h,TREM-1 returned to normal gradually.2. The prokaryotic expression vector of mouse TREM-1 fusion protein was successfully constructed and achieved stable expression and purification.A large number of recombinant mouse TREM-1 fusion protein was expressed and purified and was identified to be the target protein.3. Fusion protein TREM-1 effectively alleviated the inflammatory response in mice, and reduced the damage in pancreas and lung tissue,and enhanced the survival rate.Animal experiments confirmed that TREM-1 as a molecular therapeutic targets for the treatment of SAP may be a new choice.Summary:The study demonstrated that TREM-1 played an important role in the process of severe acute pancreatitis in mice.Preliminary studies confirmed the TREM-1 expression in SAP mice.Mouse TREM-1 fusion protein prokaryotic expression vector was successfully constructed and was used in therapeutic intervention in SAP mice. Responses by enzyme-linked immunosorbent assay to detect serum TREM-1, CRP, TNF-1 and other inflammatory factors, as well as to observe the effect of pathological changes in the organization, which verified the TREM-1 as a molecular therapeutic targets for the treatment of SAP. The detection of sTREM-1 levels in patients with SAP will be valuable in clinic.This study laid a foundation for finding the natural ligands of TREM-1.It was also useful for us to understand the pathogenesis and provides a new way for the treatment of SAP.