Experimental Research On Between The Expression Of β-catenin,Tcf-4,LZM And Proliferation And Differentiation Of Intestinal Stem Cell In Intestinal Mucosa During Severe Abdominal Infection

Objective To observe the time-dependent expressions of as well as distributionchanges and features of beta catenin(β-cat), T cell factor 4 (Tcf-4) and lysozyme(LZM) in the rat intestinal mucosa during severe abdominal infection. Meanwhile, thepossible biological effects ofβ-cat, genetic transcription of Tcf-4 and the networkregulating mechanism of translational level on proliferation and development ofintestinal epithelial stem cells were explored.Methods Totally 40 healthy adult Wistar rats, weighed (250±20) g, wererandomly divided into 4 group with 10 rats in each group:the control group (forsimple laparotomy only), the 12 h postinfection group, the 24 h postinfection groupand the 48 h postinfection group. Cecal ligation plus puncture (CLP) was used toestablish severe abdominal infection models, which were then killed at postoperative12 h, 24 h and 48 h respectively. About 4 cm ileal tissues which 10 cm from theileocecal junction were harvested for test as the following procedures. About 2 cmharvested ileal tissues were fixed in 10％neutral formaldehyde solution for 2 h, whichwere then used for detection ofβ-cat, T cell factor 4 (Tcf-4) and lysozyme (LZM)after hematoxylin and eosin (H&E) staining and immunohistochemistry. The other 2cm ileal tissues were promptly put into nitrogen canister for preservation at thetemperature of -70℃, which were then used for detection ofβ-cat and Tcf-4 after RT-PCR. The experimental data were presented in the format of mean±standarddeviation ((?)±s). The software package SPSS 10.0 and variance analysis of completerandom design (ANOVA) were used for statistical analysis.Results (1) Morphological study after H & E staining demonstrated that rats ofthe control group had complete intestinal mucosa layer structures and well-arrangedintestinal villi under the light microscope, while rats from the experimental groupshad interstitial edema and vascular engorgement in intestinal mucosa and submucouslayer, neutrophil infiltration in the mucous layer, blood capillary engorgement ofintestinal villi, shedding of mucous epithelium in some areas and obviousinflammatory exudation in serous membrane layer. (2) Reverse transcriptionpolymerase chain reaction (RT-PCR) was used to amplifyβ-cat and Tcf-4 genes fromdifferent stages of postinfection. The results showed that at 12 h postinfection mRNAlevel quickly increases (0.74±0.10 vs 0.52±0.06, P＜0.01; 0.21±0.01 vs 0.19±0.01,P＜0.01), compared to the normal control. At 24 h postinfection, the level of mRNAreached its peak level and was (0.90±0.09 vs 0.52±0.06, P＜0.01; 0.28±0.02 vs0.19±0.01, P＜0.01), as compared to the control group. Since then, the expressiongradually decreased, while at 48 h postinfection the expression was still significantlyhigher than the normal control (0.80±0.09 vs 0.52±0.06, P＜0.01; 0.20±0.01 vs0.19±0.01, P＜0.05). (3) The results of the immunohistochemistry showed that bycomparison ofβ-cat, Tcf-4 and LZM expressions in the intestinal crypt between pre-and post- operation, it was found that normal tissue has weak expression ofβ-cat,Tcf-4 and LZM, while obviously increased expressions at postoperative 12 h(145.70±8.31 vs 134.80±10.56, P＜0.05; 73.20±11.23 vs 49.00±10.45, P＜0.01;74.70±7.32 vs 65.80±5.83, P＜0.05). Among the three experimental groups, the 24 hpostinfection group had the strongest expressions (178.20±10.51 vs 134.80±10.56,P＜0.01; 112.00±11.60 vs 49.00±10.45, P＜0.01; 83.70±8.27 vs 65.80±5.83, P＜0.01),compared to the blank control. There was a time-dependent decrease of theirexpressions. At the postoperative 48 h, there were still expressions in the intestinalcrypt (146.60±9.92 vs 134.80±10.56, P＜0.05; 63.70±11.04 vs 49.00±10.45, P＜0.01;76.70±7.75 vs 65.80±5.83, P＜0.01) comparing to the blank control. By statisticsanalysis, the expressions of beta catein, Tcf-4 and LZM had significant differencesbetween their preoperative levels and postoperative levels. Conclusions From time-dependent expressions ofβ-cat, Tcf-4 and LZM in theintestinal mucosa tissues as well as their distributing changes and features, theexpressions ofβ-cat, Tcf-4 and LZM in the intestinal mucosa tissues may closelycorrelated with the proliferation and differentiation of intestinal mocosa stem cells.(1) It suggested thatβ-catenin might be related with the proliferation anddifferentiation of intestinal stem cell during Severe abdominal infection, and plays animportant role in damage and repair of entetic mucosa. Expression of beta catenin inthe intestinal tissues increases and the increase is quickly in the primary stage afterimpairments to the intestinal mocosa epithelium occurs in the cases of Wistar ratsevere abdominal infection, which may be closely related to Wnt/beta-cateninpathway and phosphorylation/dephosphorylation regulation of oxyphenylaminopropi-onic acid that leads to increased proliferation of intestinal epithelial stem cells,therefore, repair of impaired intestinal mucosa.(2) Tcf-4 may, to some extent, mark the proliferating state of intestinal epithelialstem cells. Therefore, test for Tcf-4 expression changes can demonstrate the repair ofthe intestinal mucosa epithelium.(3) Expression of LZM in Paneth cells can be taken as the marker indicating theproliferation and differentiation of intestinal epithelium stem cells.