Studies On The Expression Of Trypase And Chymase In Anaphylactic Reaction Death And Sudden Coronary Death Disease And Its Significance In Forensic Medicine

BACKGROUNDAn allergic reaction is typical of type Ⅰ allergic reaction, is allergic reactions of the most urgent, the most critical illness of the disease. Although allergic disease has been the hot spot of the clinical medical research, scholars at home and abroad and the pathogenesis, clinical diagnosis and clinical treatment, do a lot of research. But in the forensic practice, allergic reaction death nonspecific body often pathology change, diagnosis often according to allergy history, clinical manifestations and nonspecific morphological change (such as pulmonary edema, organ passive congestion eosinophilic infiltrate), and exclude injury, choking and poisoning and other factors can draw the conclusion, so to bring a lot of difficult forensic identification of the cause of death. And in the forensic identification for other reasons the sudden cardiac death (such as coronary heart disease death), in typical cases, also often be suspected allergic reaction death.Mast cells is human anaphylaxis starting cells, mast cells after activation can release a large amounts of promote inflammation, Such as trypase, chymase. glycoprotein, histamine. twelve of si lane and cell factor.Tryptase, almost released exclusively by tissue mast cells,is a neutral serine protease stored in mast cell granules, the most abundant type of media, accounting for20%of the total protein in mast cells. Chymase is another protease released exclusively by tissue mast cells, accounting for3%of the total protein in mast cells.it can be secreted into the extracellular matrix in the formation of taking off the planet when under the condition of ischemia, injury and other circumstances.In recent years, research shows that tryptase and chymase molecular weight more than other cause an allergic reaction medium, and the physical and chemical properties stable, therefore, tryptase and chymase is considered mast cells take off an important symbol of the particles. Forensic identification of death is allergic to relatively reliable index. However, tryptase heighten doesn’t necessarily mean that allergic reaction response, according to foreign reports in the literature, a case of the death of allergic patients, in its blood tryptase as high as106ng/mL. In recent years, some scholars found that atherosclerosis, inject heroin death, trauma cause death, and sudden death syndrome in infant body in the effort of tryptase also have different degrees of elevated. Recent studies have found that mast cells not only involved in allergic reactions, but also play an important role in the initial immunization and immune regulation.In patients with cardiovascular disease, mast cells can induce the apoptosis of cardiac myocyte, the proliferation non-myocardial cells including fibroblasts, vascular endothelial cells, and a strong and effective promotion of angiogenesis, which plays an important role in cardiovascular disease.SCD often happened quickly,, and the visual observation and regular histological section of H.E. stainning can be difficult to find significant pathological morphology change.Organization also have more tryptase express, which makes for classes allergic dead become more complex, survey the allergy of the mast cells reaction, then become determineallergic shock or get rid of other response of cause of the death reaction extremely distinguish the means importantly.OBJECTIVEAt present studies about the mast cells of heart,lung and Tryptase levels of serum both in allergic shock and coronary heart disease death reported at home and abroad are rare. For this reason, we applied immunohistochemical and image analysis technique to detect the expression of Tryptase and Chymase in allergic shock and coronary heart disease death qualitatively and semi-quantitatively and Analysis the differences of tryptase levels of serum to explore the relationship between these two kinds of protein and the allergic shock death and sudden death of coronary heart,and its clinical significance in allergic shock, which provide the scientific basis for forensic pathology diagnosisMATERIALS AND METHODS1.Semi-quantitative judgement of EosinophilsThe degree of allergic reaction was evalued by caculating the the number of eosinophils including specimens of throat, lung and intestine froml2cases of allergic reaction death people under the200-fold objective. Semi-quantitative judgement as follows:negative(no eosinophils at all) or weak (1+, the number of eosinophils<20) was defined as low degree of allergic reaction; moderate (++,20≤the number of eosinophils<40) or strong (+++, the number of eosinophils>40) as high degree of allergic reaction.2.Quantitative expression of Trypase and Chymase protein in heart and lung tissue of different groupsImmunohistochemistry SP methods were performd to detect the expression of Trypase and Chymase protein in heart and lung tissues of12cases of allergic reaction death,20cases of sudden coronary death,20cases of coronary atherosclerosis, but died because of non-cardiac disease and20patients without significant coronary atherosclerosis and cause of death was non-cardiac disease deaths. The Optical density(OD) of Trypase and Chymase protein was assessed quantitatively to analysis the two protein expression variation of heart and lung tissues which was from different cause of death by using ImagePro Plus image analysis softwore.3.Determination of IgE and tryptase in the serum of corpsesThe data of detected corpse serumn and the tryptase content including21cases of allergic reactions to death,26cases of death from cardiovascular disease and14cases of non-cardiovascular disease mortality were collected to analysis the variation of IgE and tryptase in the corpses’ serum of different causes of death.4.Statistical analysis The experimental data is expressed as mean±standard deviation((x±s)) or median [minimum-maximum persons](M [Min-Max). All statistical analysis were carried out using SPSS13.0satistical software package. Normality distribution were analyzed using the Shapiro-wilk normality test. For normal distribution of data, homogeneity of variance analysis was used to further examine the overall variance of the corresponding samples; if variances are heterogeneous, Dunnett’s T3test were used for multiple comparisons between groups, if not, LSD test were used. Between the two groups used the two independent-sample t-test.For skewed distribution data, multiple groups were compared using a number of independent samples of non-parametric tests (K-Independent Ssmples Test) between the two groups two independent samples of non-parametric tests (Mann-Whitney U) was used. Non-parametric tests using χ2test. A2-sided P<0.05was considered statistically significant in all the analysis.RESULTS1.Eosinophils in different tissues of allergic reaction deathThe eosinophil number in the three organs including throat, lungs and intestines of allergic reaction death group were different, and the difference were statistically significant (χ2=6.623, P=0.036), and the number of eosinophilic granulocyte cell infiltration in lung and intestinal was higher than that in throat.2.Expression of Trypase and Chymase protein in lung tissue(1)Optical density(OD) value of Chymase in negative control group, Coronary heart disease(CHD)group, sudden coronary death(SCD)group,allergic reaction group respectively are0.019±0.003,0.049±0.004,0.049±0.004,0.049±0.004. OD value of Trypase showed difference among these groups(P=309.566, P=0.000), OD value in SCD group, CHD group and allergic reaction group is higher than that in negative control group respectively(P=0.000), there were no difference between SCD group and CHD group (P=0.942),there were no difference between CHD group and allergic reaction group(P=0.965),there were no difference between SCD group and allergic reaction group(P=0.985).(2)Optical density(OD) value of Trypase in negative control group, Coronary heart disease(CHD)group, sudden coronary death(SCD)group, allergic reaction group respectively are0.045±0.048,0.171±0.020,0.220±0.019,0.289±0.031, OD value of Chymase showed difference among these groups(F=174.876,P=0.000), it showed significant difference between each two groups (P=0.000).3.Expression of Trypase protein in heart tissue(1)Trypase OD value of left ventricular wall in negative control group, Coronary heart disease(CHD)group, sudden coronary death(SCD)group, allergic reaction group were increased and repectively as follows:0.019±0.008,0.035±0.005,0.049±0.007,0.107±0.358, it showed difference among these four groups (F=290.122, P=0.000), and the difference were significant between each two groups (P=0.000).OD value in the degree of stenosis of the left coronary artery is less than level Ⅲ group, equal to the grade Ⅲ group, and greater than or equal to grade Ⅳ group respectively were0.025±0.010,0.070±0.030,0.088±0.031, it showed difference among these groups(F=52.624,P=0.000). OD value were higher in the degree of stenosis of the left coronary artery which is equal to the grade Ⅲ group and greater than or equal to grade Ⅳ group than that in less than level Ⅲ group(P=0.000, P=0.000), it showed no significance between the group qual to the grade Ⅲ and equal to grade Ⅳ(P=0.212).(2)Trypase OD value of left anterior descending artery in negative control group. Coronary heart disease(CHD)group, sudden coronary death(SCD)group, allergic reaction group were repectively as follows:0.030±0.010,0.047±0.005,0.064±0.009,0.114±0.035. It showed difference among these four groups(F=187.593, P=0.000), and the difference were significant between each two groups(P=0.000).OD value were respectively were0.036±0.012,0.084±0.027,0.094±0.030in degree of stenosis of left coronary artery less than level Ⅲ group, equal to the grade Ⅲ group, and greater than or equal to grade Ⅳ group(F=56.350, P=0.000), OD value in the degree of stenosis of left coronary artery equal to the grade Ⅲ group, and greater than or equal to grade Ⅳ group were respectively higher than that in less than level Ⅲ group(P=0.000, P=0.000), OD value shoowed no difference between the degree of stenosis of left coronary artery equal to the grade Ⅲ group, and greater than or equal to grade IV group(P=0.592).(3) Trypase OD value showed difference among these four groups(F=258.455, P=0.000),It showed difference between lung and left ventricular wall (F=1047.839, P=0.000),Mutual effect between group and organ showed difference (F=120.774P=0.000)4. Analysis the differences of IgE and tryptase levels of serum in different groups(1)IgE levels of serum in allergic reaction group, cardiovascular disease group and non-cardiovascular disease group were104.000[27.200～915.700],116.500[2.400-278.000] and6.150[2.200～20.000] respectively, difference were statistically signifi--cant among groups(χ2=16.000,P=0.000). According to the average rank times further concluded, the blood IgE content allergic reactions is the highest group, cardiovascular disease group is the second smallest, and the lowest group is non-cardiovascular disease group.(2)Tryptase levels of serum in allergic reaction group, cardiovascular disease group and non-cardiovascular disease group were80.700[2.290～667.600],15.374[0.000～36.600] and6.150[2.100～10.600] respectively, difference were statistically significant among groups(χ2=26.667,P=0.000). According to the average rank times further concluded, the blood IgE content allergic reactions is the highest group, cardiovascular disease group is the second smallest, and the lowest group is non-cardiovascular disease group..CONCLUSIONS1. The increase of the number of eosinophilic infiltration has an important value in the diagnosis of anaphylaxis reaction death.2.Compared with negative control group, Chymase protein were higher in Coronary heart disease(CHD)group, sudden coronary death(SCD)group, anaphylaxis reaction death group, however, Chymase only account for3%of the total protein in mast cells, and the expression is less. So it showed no significant difference among Coronary heart disease(CHD)group, sudden coronary death(SCD)group, and anaphylaxis reaction death group. 3.Trypase OD value of heart tissues in negative control group. Coronary heart disease(CHD)group, sudden coronary death(SCD)group, anaphylaxis reaction death group were increased, and the differnce were significantly between each two groups; Trypase OD value of lung tissues in negative control group, Coronary heart disease(CHD)group, sudden coronary death(SCD)group,anaphylaxis reaction death group also increased, and the differnce were significantly between each two groups. Tryptase level in body serum showed statistical significance between allergic reaction group and cardiovascular disease group.Tryptase can provides new ways for anaphylaxis reaction death diagnosis.4.IgE level body serum showed no statistical significance between allergic reaction group and cardiovascular disease group, which indicated that we should reference to other indicators in the dignosis of allergic reaction death.