肠三叶因子对新生大鼠坏死性小肠结肠炎模型的作用研究

发布时间：2018-06-01 23:44

本文选题：肠三叶因子 + 新生儿坏死性小肠结肠炎　；参考：《武汉大学》2009年博士论文

【摘要】：目的：研究肠三叶因子(ITF)对坏死性小肠结肠炎(NEc)新生大鼠的肠粘膜中促炎细胞因子(IL-1β,IL-6)和抗炎细胞因子(IL-10)含量的影响,以及对NEC新生大鼠中核因子-κB(NF-κB)的活化及胞内分布规律；观察MAPK通路在新生大鼠NEC模型发病中的作用,探讨ITF对NEC保护作用机制。方法：50只新生鼠随机分为5组(A、B、C、D、E),每组10只：A组为正常对照组,未进行缺氧、低温及注射用药；B组为正常组予以皮下注射ITF0.2mg(0.2m1)；C组为NEC模型后未进行注射用药；D组为NEC模型后予以皮下注射生理盐0.2ml；E组为NEC模型后予以皮下注射ITF0.2mg(0.2m1)。并于NEC模型后第4天处死并取回盲部肠组织1-2cm固定、包埋、切片、HE染色拍照观察组织学变化及免疫组化观察NF-κB及MAPK通路的表达,其它肠组织制备组织匀浆,取上清液检测IL-1p、IL-6、IL-10含量。结果：NEC模型后给药E组病理组织学改变积分范围从0-2分,与正常对照组相比,粘膜下层或固有层有轻度分离,而模型组C、D组病理组织学改变积分范围从2-4分,有粘膜下层或固有层有重度分离,粘膜下层和肌层有严重水肿,该区的绒毛崩解分离和绒毛的缺血坏死。NEC模型后给药E组肠粘膜组织匀浆IL-1β含量明显低于模型组C、D(P0.01)而与正常组A、B组无显著性差异(P0.05)。NEC模型后给药E组肠粘膜组织匀浆IL-6含量明显低于模型组C、D(P0.01)而与正常组A、B组无显著性差异(P0.05)。E组IL-10含量明显高于C、D组(P0.01)和A、B组(P0.01)；新生大鼠NEC模型中肠上皮细胞NF-κB(p65)阳性表达,阳性颗粒数和染色强度明显高于阴性对照和正常组；新生大鼠NEC模型中皮下注射ITF后肠组织NF-κB9(p65)呈弱阳性表达,与免疫组化染色阴性对照和正常组相比呈阳性,但与NEC模型组NF-KB(p65)阳性表达相比明显减弱；新生大鼠NEC模型后MAPK通路中MEK/ERK/Elk-1, JNK/c-Jun以及p38均发生磷酸化激活；予以ITF治疗新生大鼠NEC模型中发现,JNK/c-Jun和p38表达较NEC模型组无明显升高,而MEK/ERK/Elk-1通路则显著激活。结论：1)ITF可能是抑制促炎细胞因子(IL-1β,IL-6)的表达和促进抗炎细胞因子(IL-10)的合成,并通过抑制NF-κB信号通路,从而抑制其代谢合成产物的生成,减少细胞炎症因子的升高,进而减轻小肠结肠组织炎症反应,达到了保护肠粘膜的作用；2)新生大鼠NEC模型后MAPK通路中MEK/ERK/Elk-1, JNK/c-Jun以及p38均发生磷酸化激活,该结果提示MAPK参与NEC发病过程；予以ITF治疗新生大鼠NEC模型中发现,JNK/c-Jun和p38表达较NEC模型组无明显升高,而MEK/ERK/Elk-1通路则显著激活,该结果提示了ITF可能是通过MAPK中MEK/ERK/Elk-1通路发挥对新生NEC大鼠的治疗作用
[Abstract]:Objective: to study the effects of intestinal trefoil factor (ITF) on the contents of proinflammatory cytokine (IL-1 尾 -IL-6) and anti-inflammatory cytokine (IL-10) in the intestinal mucosa of nec rats, and on the activation and intracellular distribution of nuclear factor- 魏 B NF- 魏 B in neonatal rats with NEC. To observe the role of MAPK pathway in the pathogenesis of NEC in neonatal rats and to explore the protective mechanism of ITF on NEC. Methods Fifty newborn mice were randomly divided into 5 groups, 10 in each group as normal control group without hypoxia. The hypothermia group and the control group were subcutaneously injected with ITF 0.2 mg / m ~ (-1) NEC model. Group D was treated by subcutaneous injection of physiological salt 0.2ml / ml NEC model and then subcutaneous injection of ITF _ (0.2) mg ~ (-1) ~ (0.2) mg ~ (-1) after subcutaneous injection of ITF _ (0.2) mg ~ (-1) ~ (-1). On the 4th day after NEC model, the 1-2cm was fixed and embedded in the intestinal tissue of the blind part. The histological changes and the expression of NF- 魏 B and MAPK pathway were observed by HE staining and immunohistochemistry. The homogenate of other intestinal tissues was prepared. The supernatant was used to detect the level of IL-6 and IL-10 in the supernatant. Results the score of histopathological changes in group E was 0-2 points after the administration of the control group. Compared with the normal control group, the submucous layer or lamina propria was slightly separated, while the score of histopathological changes in group C was 2-4 points. The submucosa or lamina propria has severe separation, and the submucous and muscular layers have severe edema. The content of IL-1 尾 in intestinal mucosa homogenate of group E was significantly lower than that of group C (P < 0.01), but there was no significant difference between group A and group A (P 0.05). The content of IL-6 in plasma was significantly lower than that in model group (P 0.01), but there was no significant difference between group A and group A (P 0.05). The content of IL-10 in group E was significantly higher than that in group C (D) and group A (P 0.01), and the positive expression of NF- 魏 Bp65) in intestinal epithelial cells in neonatal rat model of NEC. The number and intensity of positive granules were significantly higher than those of negative control and normal group, and the expression of NF- 魏 B9 p65 in intestinal tissue after subcutaneous injection of ITF was weakly positive in neonatal rat model of NEC, and was positive compared with that of negative control group and normal group by immunohistochemical staining. However, compared with the NEC model group, the expression of NF-KBP p65) was significantly decreased, the expression of MEK / ERK / Elk-1, JNK/c-Jun and p38 was activated in the MAPK pathway of neonatal rats after NEC model, and the expression of JNKP / c-Jun and p38 in NEC model of neonatal rats treated with ITF was not significantly higher than that in NEC model group. The MEK/ERK/Elk-1 pathway was significantly activated. Conclusion 1 / 1 ITF may inhibit the expression of pro-inflammatory cytokine IL-1 尾 -IL-6 and promote the synthesis of anti-inflammatory cytokine (IL-10). It may inhibit the production of its metabolites and decrease the increase of cytokines by inhibiting the signal pathway of NF- 魏 B. Then the inflammatory reaction of intestinal colon tissue was alleviated and the intestinal mucosa was protected. (2) phosphorylation of MEK / ERK / Elk-1, JNK/c-Jun and p38 in MAPK pathway was observed in neonatal rat NEC model. The results suggested that MAPK was involved in the pathogenesis of NEC. The expression of JNKc-Jun and p38 in neonatal rats treated with ITF was not significantly higher than that in NEC model group, but the MEK/ERK/Elk-1 pathway was significantly activated. The results suggest that ITF may play a therapeutic role in neonatal NEC rats through MEK/ERK/Elk-1 pathway in MAPK.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R725.7;R-332

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