Research Of Identification Of The Eggs Dead Or Alive Which Were Extracted From The Colonic Mucosa Of Mice Infected With Schistosoma Japonicum And Antigen Detection

[Objective] The purpose of this study is to examine antioxidase levels in Schistosoma japonicum (Sj) eggs of different development durationdevelopmental stages, and evaluate if antioxidase staining can be used to identify the viability of eggs trapped in human tissue are dead or alive. Mice were randomly divided into groups of50and90-day days after infection, and those30and90-day days after treatment, with viable Sj in the firstformer two groups and no viable Sj in the lastlatter two groups, respectively. Antioxidase staining the free. Free eggs separated from the liver and colonic mucousmucosa of different groups were antioxidant-stained with substrates3,3’-Dimethylbenzidine and hydrogenperoxide as its substrates and detecting (the staining conditions, then comparative observing were also studied). Then the enzyme levels of different groups were compared by their coloration and the distribution thereof in order to evaluate its utility. We had adopted Superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), peroxidase (POD), catalase (CAT) activity assay kits to determine the type and level of antioxidant enzymes within the eggs.[Results] In contrast to the eggs not dyed, the positive DAB-treated ones’dark brownish-yellow reachedcoloration, generated by the positive reaction, became the most visibleobvious when the free eggs were stainingstained at room temperature and treated by oscillation for30minutes. No coloration were seen oncein the eggs boiled with DAB substrates for30minutes. The positive rates of the different four groups reached92.34%.46.03%、13.68%and0.00%.%, respectively. The extents of positive degree showedreactions were manifested in that immature eggs presented dark brownish-yellow, mature eggs were deep yellow and located in myracidium body, the currenteggs recently subjected to degradation eggs were partlypartially dyed in which (most of the dyed were immature eggs), and the lateeggs early subjected to degradation eggs and shell appearedthe shells showed no positive reaction.[Conclusions] The conclusion is that the viable Sj eggs are rich in antioxidase, particularly inthe immature eggsones, while the enzyme levels fade awaydecline as eggs develop from maturity to death. So the results suggest that antioxidase staining of Sj eggs can be a marker to discriminatefor the discrimination of viable orand dead eggs. To explore the method to separate the eggs from the tissues, and to improve the effect of the antioxidant enzyme staining which is to distinguish the eggs within the biopsy dead or alive.[Methods] Use six kinds of methods which are tableting, shearing, ultrasonic, chemical (5%NaOH) digestion, enzyme (0.5%trypsin and1%pepsin) digestion to deal with the simulated biopsy tissue blocks of mouse intestinal mucosa, observe the eggs to free the tissues and the effect of DAB staining. Compared with the other processing methods, trypsin enzyme digestion is the best to free the eggs and to obtain more satisfactory staining effect of antioxidant enzymes. Tableting and shearing were ineffective to free the eggs,.The ultrasonic method led to rupture and loss of part of the eggs. Both NaOH and pepsin digestion method mainly affects the staining effect of antioxidant enzymes. Trypsin enzyme digestion can free most eggs from the intestinal mucosa and get a good antioxidant enzymes staining results, however, further research to free all the eggs is still needed. 【Objective】 Detect the soluble antigen which is secreted by the Sjmiracidia from the colonic mucosa of mice infected with Sj and exudativeinto the host tissue in order to provide the experimental basis for developinga new diagnostic method compensating the deficiency for the biopsy ofintestinal mucosa.【Methods】Prepare rabbit and mouse anti-6‘/eggspolyclonal antibody for exploring the feasibility of enzymeimmunohistochemistry and immunofluorescence to detect the antigenof Sj eggs of the colonic mucosa of mice infected with^/.Establish indirectsandwich ELISA essay and explore the best conditions, then take the colonicmucosa simulated biopsy tissue blocks of normal mice,90d mice atferinfection,1M and3M mice atfer treatment, separate the containing eggs andno eggs tissues through the microscope, comparatively detect thesupernatant by the ultrasound and the homogenate processing. Based on theOD value2.1times of negative reference control, it can be identiifed aspositive. Mouse colon paraffin sections were detected by indirect enzymeimmunohistochemistry method, a brown yellow mark observed in themucosa was sentenced to positive. Make the statistical analysis and effectsassessment based on the positive rate detected by the two methods.【Results】 The enzyme immunohistochemistry method had no positiveresult. Egg antigen was detected through immunofluorescence assay, but theeffect was not ideal. The indirect sandwich ELISA results show: the antigenpositive rates between the containing eggs and not within the mucosa atferultrasound treatment were58.97%and60.00%in group A (90d mice atferinfection)； were37.50%and25.00%in group B(treatment for1M atferinfection for50d);were13.89%and0.00%in group C(treatment for3Matfer infection for50d).There was no positive result in group D(normalgroup).The antigen positive rates between them atfer homogenate processingwere45.00%and00.0%in group A. There were no positive result in groupB,C and D.【Conclusions】 The indirect sandwich ELISA to detect eggantigen in the mouse colonic mucosa has a good diagnosis and assessment ofthe efficacy of value, but need to be improved the detection sensitivity.