Expression Of Tryptase,Chymase And PAR-2 In Anaphylactic Shock Mice

Objective In this experiment,KunMing male mice as experimental subjects,OVA as allergen,by the way of intravenous injection of ovalbumin in mice to establish the animal model of anaphylactic shock.Through the study of expression and their correlation of tryptase,chymotrypsin in peripheral blood serum and proteinase activated receptor 2 mRNA expression in blood to provides new ideas to the treatment of allergic shock in clincal.Method CL male Kunming mice provided by Animal Center of,16 ~ 25 g.Sixty KM male mice were randomly divided into two groups: the experimental(n=40)and the control(n=20).Because the difficultion of taking samples is larger after the experimental mice died.In order to ensure adequate samples,the number of mice in the experimental group is two times higher than that of the control group,failure rate of obtaining specimen is 50%.Mice were fed in different chests taking food and drink freely,before the start of the experiment mice were fed 1 weeks in order to adapt to the environment.The experimental was immunized by intraperitoneal injection of allergen0.25 ml at first day and then duplicated after a week.Allergic shock was induced by tail vein injection of allergen 0.5ml after three weeks later.The control were injected equal amount of PBS buffer to replace.Observed the clinical manifestations of two group mice,collected the blood by the way of enucleating the mouse eyeball(sodium citrate anticoagulation tube).ELISA was used to detect the expression of tryptase and chymase in mice serum.RT-PCR was used to detect the relative expression of PAR-2mRNA in mice blood.After spinal transection,the animals were dissected and obtained the lung,stomach,intestinal tissue and fixed in 40% phosphate buffered formalin.Tissues were sliced and HE stained for histological examination.Result1.The clinical symptoms observation records After tail intravenous injection ofallergen,experimental groups of mice appeared different degree of forelimb scratching nose similar to the action of cleaning face action in 8 minutes and were very excited in the cages.About 25 minutes later,the mice of the experimental group is creeping motionless,reduce the volume of activities,listlessness,eyes narrowed.Most of the mice recovery to normal state about 50 minutes later.The rest of the mice’s symptoms worsened and gradually appeared motionless,sluggish performance etc.In severe cases,there are performance just like the shortness of breath,convulsions and even incontinence.A few mice died in about an hour.Observation of the normal control didn’t appear the above symptoms.2.Histopathological examination Under optical microscope we can see edema appeared around the small blood vessels of the lung,hemorrhage,widened alveolar septum and destruction of lung tissue structure in the experimental group of mice.Gastrointestinal tissue sections showed gastrointestinal mucosal edema,the gap widened.The normal control group of mice’s organs showed no obvious histological abnormalities under optical microscope.3.RT-PCR results In the experimental group of mice,the relative expression of blood PAR-2 mRNA is(3.372±0.28).In the control group the relative expression of blood PAR-2 mRNA is(0.759±0.062).In the comparison of the data of two groups,the relative expression of PAR-2 mRNA in the experimental was significant higher than the control,P<0.01.4.ELISA results The expression of serum tryptase in experimental group was(72.378±8.018ug/l),and the expression of serum tryptase in control group was(20.265±1.953ug/l).The expression of serum tryptase was significantly increased as compared with the control group,P < 0.01.The expression of serum chymase in sensitized group(16.186±0.959ug/l)significantly decreased as compared with the expression of serum chymase of the control group(34.905±3.972ug/l),P < 0.01.5.The relativity on the expression level of serum tryptase,chymase and the relative expression of PAR-2 mRNA There was a significantly positive correlation between the level of serum tryptase and the relative expression of PAR-2 mRNA,r=0.837,P<0.01.The expression of serum chymase was negatively related to the relative expression of PAR-2 mRNA,r=-0.762,P<0.01.The expression of serum tryptase was negatively related to the chymase,r=-0.647,P<0.01.There were no significant relativity on the expression level of serum tryptase,chymase and the relative expression of PAR-2 mRNA.Conclusion1.KunMing male mice animal model of anaphylactic shock is established by intra-peritoneal and tail vein injection OVA.2.As the only known receptor of tryptase,PAR-2 may participate the process of anaphylactic shock,and play a positive regulatory role in the pathogenesis of anaphylactic shock.