Preliminary Study On Protective Effects Of Anti-adhesion Molecule CD146 Monoclonal Antibody AA98 In Septic Mice

Objectives To explore protective effects of anti-adhesion molecule CD146 monoclonal antibody AA98 in septic mice, to explore a new comprehensive therapy on sepsis by combining anti-endotoxin monocloanal antibody and/or anti-adhesion molecule monocloanal antibody with operation.Methods 1. The reformed laboratory mode of sepsis was established by excision of cecum and saline peritoneal lavage when the cecal ligation and punction was followed at 8 hours. 2. The anti-CD 146 monoclonal antibody AA98 were prepared with the mice that immunized by hybridoma clone AA98 that has been made in our laboratory. purified monclonal antibody AA98 was accepted by Protein A affinity chromatography. The Western -Blotting was used to detect specificity of the antibodies binding to CD146, ELISA was used to detect titer of antibody AA98, 10% SDS-PAGE electrophoresis was also used to detect its purity . 3. All animals were randomly divided into 4 groups(32/group):A group (control group), B group (anti-CD 146 monoclonal antibody AA98 group), C group (anti-endotoxin monoclonal antibody M2b group) and D group (AA98 and M2b). AA98 were injected through tail vein at 2 hours and 6 hours after operation, and M2b were injected through tail vein at 2 hours after operation. WBC ,LPS and TNF-αlevel in blood were assayed at 12 and 24 hours after operation, Lung and liver pathological changes were also observed. the mortality rate at 24 , 48, 72 and 96 hours after operation were recorded.Results 1. The reformed laboratory mode of sepsis corresponded to operate on mice whose cecum had been ligated and punctured,so dead time of animal was delayed,and animal mortality rate was low in early period;but animal mortality at 72 hours after operation in modified group reached to 80%;WBC, blood-serum levels of endotoxin and TNF-αin modified group was significantly higher than in sham group (P<0.05) .Meanwhile, Lung and liver showed secondum pathological changes in modified group at 24 hours after operation. 2. The monoclonal antibody AA98 that were made in our laboratory can specially bind with CD146, and its titer and density was 1:20000 and 1.804mg/ml respectively; purity of monclonal antibody AA98 that was accepted by Protein A affinity chromatography was higher than that of certain antibody made in China. 3. Animal mortality at 48 hours and 72 hours in the control group was 60% and 80% respectively. If animal were injected anti-endotoxin and/or anti-adhesion monocloanal antibody after operation, mortality at 48 hours and 72 hours was significantly lower than in the control group(P<0.05),meanwhile, WBC, blood-serum levels of endotoxin and TNF-αat 12 hours and 24 hours after operation in these groups were also significantly lower than in control group (P<0.05) ,and these data in combining(M2b+AA98) group were also lower than in anti-endotoxin or anti-adhesion molecule monocloanal antibody group.Lung and liver pathological changes at 24 hours after operation in these groups were significantly slighter than in control group.Conclusion 1. Animals were operated after the cecum was ligated and punctured,their dead time were delayed and mortality(at 24 hours) in the early phase were degraded,which will be easy to study on comprehensive therapy.of sepsis.. 2. Animals, on basis of operation operative treatment, were injected anti-adhesion monocloanal antibody AA98, their systemic inflammatory response was relieved significantly,and pathological impairment of vital organ was abated remarkably, their dead time were delayed significantly and mortality(at 48 hours and 72 hours) in the later phase were degraded. So anti-adhesion molecule monocloanal antibody AA98 had protective effecs on septic mice.3.If Animals were injected anti-endotoxin and anti-adhesion molecule monocloanal antibody ,their mortality in the later phase was significantly lower than animal that was injected anti-endotoxin or anti-adhesion molecule monocloanal antibody respectly,so the therapy of combining anti-endotoxin with anti-adhesion molecule monocloanal antibody on basis of operation in the early phase should be a new therapeutic mode on sepsis.