Observe The Role Of PTEN, Lgr5 And β-catenin In Intestinal Injury And Recovery After Necrotizing Enterocolitis

Objective:To establish and evaluate NEC model as non-operative NEC patients in neonate rat. And to observe the change of the Phosphatase and Tensin Homolog deleted on Chromosome Ten (PTEN), Eucine-rich-repeat-containing G-protein-coupled Receptor 5 (Lgr5) and Cadherin Associated Protein Beta (β-catenin) in intestine injury and recovery after Necrotizing enterocolitis, in order to disccus the effect of PTEN, Lgr5 and P-catenin in intestinal ischemic injury and self-recovery for researching the pathogenesis of NEC and providing the possible idea for therapeutic approach.Part 1 Establish and Evaluate of the intestine self-recovery NEC model after intestine injuryMethod:Establish intestine self-recovery NEC model by hypoxia and cold stimulation in neonatal rat model. Newborn rats were divided into control group (A group,15) and experimental group (B group,15). Experimental group was stimulated by hypoxia and cold for 3 days beginning postnatal first day. Rats were sacrificed 24h,72h and 120h after last hypoxia and cold (B1, B2 and B3 groups) in experimental group, respectively. Control group rats are no stimulate and were sacrificed in same time (Al, A2 and A3 groups). Every group has 5 rats. Compare the weight between pre-experiment and experiment forth day. Observe the neonatal rat model during the stimulation. Find out neonatal rats whether abdominal distension, gastric retention, decline in activity, and so on. Make pathological tissue sections to understand intestinal damage and changes in villus structure by HE stain. Use Nadler standard assessment scores to evaluate intestinal injury in HE stain.Results: 1.The experimental group appearance different degrees of activity decreased, eating down, mild to moderate diarrhea, visible abdominal, gastric retention and gradually aggravate in 3 days by hypoxic and cold stimulation. These symptoms are lighten and disappearance in 72h and 120h after stimulation2. Experimental group weight gain was significantly lower than the control group, P<0.05, statistically significant.3. In Autopsy, the intestine color is ruddy, and good elasticity in control group; part of intestines visible edema, thickening and significant bleeding in B1 group; the intestine color are normal in B2 and B3 groups.4. In control group, villus structural integrity intact; mucosa and epithelial integrity and continuity; glandular arrangement rules; the organizational structure clear, active secretion vascular in propria lamina no expansion; muscle no abnormal, no inflammatory cell infiltration and ulceration. In B1 group, intestinal submucosa hyperemia and edema; inflammatory cell infiltration in lamina propria; histopathology scores were level at 2-3. B2 and B3 compared with B1 group, intestinal damage reduces; small amount of mucous membrane shedding; small inflammatory cell infiltration; histopathology scores were level at 1-2.Part 2 Observe the role of PTEN, Lgr5 andβ-catenin in Intestinal Injury and RepairMethod:Establish the NEC simulating NEC intestinal injury and self-repair in clinical by hypoxia and cold stimulation in neonatal rat model. All rats take ileocecal bowel (specific method refers part one) in 24h,72h and 120h after last hypoxia and cold. Determined PTEN, Lgr5 andβ-catenin mRNA expression levels in intestinal tissue by Realtime PCR. Comprehending the role of the PTEN, Lgr5 andβ-catenin in intestinal self-repair after intestinal injury. Analyze the relationship of the PTEN, Lgr5 andβ-catenin. Approach them possibly role in intestinal proliferation.Results:1.Compared the experimental group with control group, PTEN, Lgr5 andβ-catenin mRNA expression significantly higher (P=0.013; P=0.047; P=0.020) at 24h after last hypoxia and cold, significant deviation, statistical significance.2. At 72h and 120h after last hypoxia and cold, expression levels are not significantly different (P=0.630, P=0.864; P=0.481, P=0.353; P= 0.472, P=0.259) between experimental group and control group, no statistically significant.3. In experimental group, PTEN, Lgr5, P-catenin expression levels at 24h, is not significant difference (P=0.053; P=0.105; P=0.681), no statistically significant.4. Compared 24h with 72h and 120h in experimental group, PTEN, Lgr5 and P-catenin mRNA expression levels are significantly increased (P= 0.002, P=0.001; P=0.011, P=0.001; P-0.030, P=0.006), statistical significance.5. Compared expression levels at 72h and 120h in experimental group, are not significantly different (P=0.941; P=0.525; P=0.746), no statistically significant.Conclusion:1-Successfully establish intestine injury and recovery NEC model by Nitrogen include 6% oxygen and 4℃in neonatal rat.2. In NEC intestine injury period, PTEN, Lgr5 andβ-catenin mRNA expression levers are significantly increased at 24h after stimulation.3. However, in intestine recovery period, PTEN, Lgr5 and P-catenin mRNA expression levers are significantly decreased at 72h and 120h after stimulation. Compared the experimental group with control group, it is not statistically significant.4.β-catenin expression was increased, may be able to promote stem cell proliferation in intestine injury.5. PTEN can balance stem cells proliferation speed and stem cell number by regulating the activity ofβ-catenin.