| Objective: To explore the role of bifidobacteria on the morphology in a neonatal rat model of necrotizing enterocolitis(NEC),meanwhile,to detect the gene expression of iNOS,EGF of ileum by fluorescent quantitation PCR,and study whether the protection by bifidobacteria is relative to the suppression of up regulation iNOS and down regulation of EGF,so as to provide the molecular biological evidengce applying bifidobacteria to prevent and cure NEC in newborn.Method:1. Animal and group: The neonates which are 12 hours after birth are divided into 3 groups. Group A: Experimental group,13 rats fed with bifidobacteria and modeled. Group B: Model group,13 rats were made model;Group C: Control group,13 rats fed without any probiotic and bacteria.2. The way adding bifidobacteria: Adding viable bifidobacteria to formula milk before feeding, concentration of bifidobacteria in formula milk is 1*108cfu/ml, twice daily.3. Setting up the NEC model: The neonates within 12 hours after birth were gavage fed with a special rodent formula four times daily,and subjected to 90 sec of hypoxia twice daily (100% N2), 4℃cold exposure for 10 min during 3 consecutive days. After the last hypoxia and cold exposure, rats were gavage fed LPS 20ul(concentration 2.5mg/ml).5. Experimental method and index: Observe the weight of the neonates before and after modeled;the mortality of the three groups during modeld; ileum tissues were affixed by formaldehyde solution,imbedded in paraffin,and stained with H.E.The tissues of each group were scored according to their injury and NEC incidences were assessed; Total RNA were extracted from the ileum tissues which were conservated in -80℃,and reverse transcripted into cDNA,the gene expression difference of iNOS,EGF between each group were tested by fluorescent quantitation PCR.6. statistics method: The datas were calculated by SPSS 13.0. measurement datas were presented by mean±standard deviation(?x±s),t-test or Wilcoxon rank sum test was used for group comparison,χ2-test was used for rate comparison,it was considered significant difference with P<0.05.Results:1. After modeled,the weight of group B rats was lower after modeled[ - ( 0.23±0.59 ) g ] ,while the group A rats were lightly higher[(0.41±0.59)g], and the weight increment in group C rats were normal during the experimental time(2.45±0.34)g].2. The mortality of group A,B and C was 7.69%,15.38%,0,respectively, P>0.05 in group comparison,difference was not significant.3. The NEC incidence of group A,B and C was 38.46%(5/13),84.62%( 11/13 )å'Œ0 ( 0/13 ) , P<0.05 in group comparison,difference was significant; The ilieum pathology injury score(?x±s)of group A,B and C was 1.50±0.52,2.35±0.41,0.27±0.37,respectively;Of the total,the score of group A decreased obviously than group B,but were higher than group C, P<0.05.4. The result of gene expression tested by fluorescent quantitation PCR showed as follows: For iNOS:iNOS of group A and B were heightened significant compared with control group C,the value of A group was 2.74 times greater than group C,and the value of group B was 12.46 times as great as group C, P<0.05;The value of group B was promoted significantly when compared with group A;For EGF:Both the group A and B decreased than group C,group A was 0.52 times greater than group C,P<0.05,but group B was 0.85 times as great as group C, P>0.05;There was no significant difference between group A and B(P>0.05).Conclusions:1.The early postnatal neonates could mimic experimental NEC animal model analogous as human through fedding with a special rodent formula, hypoxia (100% N2), 4℃cold stimulus and fedding LPS of low concentration.2.The way adding bifidobacteria could decrease the weight losing.3.The way adding bifidobacteria could decrease the incidence of NEC and the injury degree of ilieum.4.The gene expression of iNOS in ileum increased when NEC. Bifidobacteria could relieve the damage to intestinal tract mucomembran,decreased the NEC incidence and the serious degree through inhibiting the activation of iNOS. |
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