【作者】 朱渝军；

【导师】 黄显凯；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 肠粘膜屏障损害导致细菌易位、肠源性感染,在多脏器功能衰竭中起重要作用。肠缺血再灌注损伤是肠粘膜屏障功能损害的常见原因。因此,加强肠粘膜的修复,预防或减轻肠屏障功能损害,对脓毒症和MODS的防治具有非常重要的意义,但迄今这方面还没有有效的治疗方法。缺血/再灌注(I/R)损伤后的肠屏障功能恢复与肠上皮细胞和肠血管内皮细胞的增殖、分化密切相关,而肠上皮细胞凋亡在肠缺血再灌注损伤中起重要作用。近年来发现的成纤维细胞因子是对肠上皮生长和功能有重要调节作用的多肽,他们通过与其受体结合激活酪氨酸激酶活性,引起受体的自磷酸化随后进入不同的信号传递途径发挥作用,而成纤维细胞因子受体在胃肠道的生物学活性却少有报道。有研究提示FGFR3参与放射损伤后的修复。而丝裂素活化蛋白激酶(MAPK)是信号从细胞表面传到细胞核内部的重要传递者。MAPK具有调节细胞生长、发育、分裂、死亡等多种细胞生理过程的作用,其亚类细胞外信号调节蛋白激酶(ERK1/2)信号传导通路是多种细胞外有丝分裂信号引起细胞增殖的共同通路,与细胞增殖有密切关系。本文拟观察野生小鼠及FGFR3增强小鼠小肠发育阶段的特点和FGFR3表达变化。另外采用小肠缺血再灌注模型,观察野生小鼠及FGFR3增强小鼠小肠粘膜缺血再灌注损伤及再生修复的特征及规律,以及肠上皮细胞增殖、凋亡的情况,检测I/R损伤后小肠FGFR3的基因表达与其对伤后肠上皮细胞ERK1/2活性的影响,从而确定FGFR3在介导肠上皮损伤修复中的作用,探讨FGFR3促进I/R损伤后肠上皮细胞修复的分子机制。最终为寻找促进肠上皮细胞损伤修复,防止肠屏障功能损害的新方法提供理论依据。第一部分FGFR3在小鼠小肠发育阶段的作用主要实验内容及方法如下:动物: Fgfr3369/+基因工程小鼠及C57BL/6J小鼠。1.组织学方法观察同窝增强突变小鼠与野生小鼠出生后第1、7、14、21、28、35天肠上皮发育过程中的组织形态学和扫描电镜观察超微结构变化。2.肠上皮增殖能力检测:用溴脱氧尿苷(BrdUrd)标记7、14、21、28、35天两组小鼠小肠上皮S期细胞(方法:按100ug/g(体重)剂量腹腔注射,2小时后取材),免疫组化(SABC法)检测其阳性表达情况。3.免疫组化(SABC法)检测两组小鼠小肠组织FGFR3的表达情况。4.提取1、7、14、21、28、35天两组小鼠小肠组织RNA,通过RT-PCR扩增后进行凝胶电泳半定量检测FGFR-3在各时相的表达水平。主要实验结果:1.突变小鼠绒毛分布密度及绒毛高度均差于同年龄野生小鼠,但隐窝深度却是相反的。2.增生的细胞主要位于肠隐窝部位,FGFR3增强突变小鼠在各时相点细胞增殖均强于野生小鼠。3.小鼠出生后1天小肠即有FGFR3表达,7-21天表达较多,35天后表达减少。免疫组化检测FGFR3表达位于肠隐窝部位,与Brdu表达信号部位相重叠。第二部分FGFR3在小鼠小肠缺血再灌注损伤修复中的作用及机制主要实验内容及方法如下:1.动物模型制备及分组:取6-8周龄小鼠,分为正常组、野生鼠I/R组和FGFR3增强小鼠I/R组,每组6只。术前12h禁食,自由饮水。用0.6%的戊巴比妥钠(60mg/kg体重)腹腔注射麻醉,暴露肠系膜上动脉(SMA),无创血管阻断剂夹闭1h后松开形成再灌注。2.标本采集、分离与储存:正常组直接活杀,另两组动物分别于松夹后1、3、6h和1、3d活杀。眶静脉窦采血,使用肝素抗凝,充分混合,以3000rpm离心10min后贮存于-20°C冰箱中,用以检测血浆D-乳酸,使用美国Thermo Labsystems Multiskan Spectrum全波长酶标仪(波长340 nm)检测。选择近端空肠,一部分肠管用4%多聚甲醛固定,石蜡包埋,切片,用以检测小肠组织PCNA表达及凋亡情况;另一部分肠管立即放入-80°C冰箱冻存,用以检测小肠组织FGFR3mRNA表达及MAPK活性检测。主要实验结果:1.肠缺血再灌注l、3、6h,肠粘膜损害明显,再灌注ld、3 d组,肠粘膜结构恢复至正常。FGFR3增强小鼠在各时间点肠粘膜损害程度均轻于野生小鼠。2.肠缺血再灌注l、3、6h,粘膜上皮细胞凋亡增加,再灌注ld、3 d组肠粘膜上皮细胞凋亡明显减少, FGFR3增强小鼠在各时间点凋亡程度均轻于野生小鼠。3.肠缺血再灌注l、3、6h,粘膜隐窝上皮细胞增殖增多,再灌注ld、3 d组粘膜隐窝上皮细胞增殖活性逐渐降低, FGFR3增强小鼠在各时间点增殖能力明显强于野生小鼠。4.两组小鼠缺血再灌注损伤后各时相点,血浆D-乳酸水平均明显高于正常对照组小鼠,并且均在再灌注1h后达到峰值,然后逐渐下降。但FGFR3增强小鼠在再灌注1、3、6h,D-乳酸水平均显著低于野生小鼠。5.两组小鼠缺血再灌注损伤后FGFR3mRNA表达明显增高,均在再灌注1h后达到第一个峰值,然后迅速下降,于3h达到低谷,再迅速升高达到第二个峰值后逐渐下降,3d仍处于较高水平。6.两组小鼠的Erk在各时间点都维持在恒定水平,而p-Erk水平在I/R损伤后明显增高,随时间推移逐渐降低,FGFR3增强小鼠缓慢降低,野生小鼠1d后降低至接近正常水平。主要结论:1. FGFR3在发育阶段促进小鼠肠隐窝形成。2. FGFR3在小鼠缺血再灌注损伤后促进肠上皮增殖,抑制凋亡。3. FGFR3促进肠上皮修复可能是通过激活ERK1/2信号通路实现的。更多还原

【Abstract】 Injury of intestinal mucosa barrier usually results in bacteria translocation and intestinal derived infection,which plays important role in MODS.Intestinal ischemia reperfusion injury is one of the most causes resulting in intestinal mucosa barrier damages.Accordingly,it is so important to improve repair of intestinal mucosa,prevent or diminish damages of intestinal mucosa barrier function and it plays important role in prevention and treatment of sepsis and MODS,however,there is no useful measurement yet.The recovery of intestinal barrier function after ischemia/reperfusion injury has close relation with proliferation and differentiation of intestinal epithelium and vascular endothelium,and apoptosis of intestinal epithelium is indispensable in intestinal ischemia reperfusion injury. Fibroblast growth factors(FGFs) are polypeptides play a key regulatory role in intestinal epithelial developmental and functions. Engagement of FGFs via FGF receptors activates their tyrosine kinase activity,leading to receptor autophosphorylation and to the phosphorylation of intracellular targets.Among growth factors involved in gastrointestinal tract development and homeostasis,FGFs have been shown to promote the proliferation and survival of intestinal cells.However,limited data is available on the biological activities of FGF receptors in the gastrointestinal tract.Recent research had proved that FGFR3 was involved in repair of intestinal epithelium after radiation injury. While mitogen-activated protein kinase(MAPK) is important transformer through which signaling pass from cell surface to nucleus. MAPK can regulate cell growth,development,differentiation,death and intercellular synchronization,and its subset ERK1/2 signaling transmission pathway is a common pathway of many extracellular mitosis signaling resulting in cell proliferation.Wild type mice and FGFR3+ mice were used to observe their characteristics of small intestinine in development phase and changes of FGFR3 expression.Moreover,patterns of small intestine ischemia reperfusion were applied to explore the features of intestinal mucosa after I/R,besides proliferation and apoptosis of intestinal epithelium.We also observed activity of intestinal epithelial ERK1/2 and FGFR3 expression after I/R injury,thus to determine the effect of FGFR3 in small intestine of mice after ischemia reperfusion injury and to explore the molecular mechanisms of FGFR3 promoting repairment of intestinal epithelium after I/R injury.Eventually,it might provide available evidence for new measurement to improve repair of intestinal epithelium and prevent intestinal mucosa barrier function damages.Part I The effect of FGFR3 in small intestine in development phaseMain methods:Animals and groups: Mice with Fgfr3369/+ (Mutants) and their littermate controls (C57BL/6J) (controls)1. Histologic morphology of small intestines of two groups at day1、7、14、21、28、35 and their ultrastructure changes.2. Proliferation of intestinal epithelium:intestinal epithelium cells in S phase were labelled at day 7、14、21、28、35 by BrdUrd(intraperitoneally 2 hours before being killed ,100ug/g),expression were determined by immunohistochemistry.3. Expression of FGFR3 in small intestine were determined by immunohistochemistry.4.Drew RNA of small intestine of two groups at day1、7、14、21、28、35,amplifaction by RT-PCR,then determined their levels through gel electrophoresis.Main results:1. Mutant mice had lower density and their villa were lower than the controls,but to the depth of crypt,the results were conversely.2.Cells in proliferation mainly located in intestine crypt,mutant mice had more proliferation than the controls at every time point.3. Expression of FGFR3 were detected at birth(day 1),the expression were maximal from day7 to day 21,decreasing rapidly thereafter to reach the relatively low at day35.And the expression of FGFR3 also located in intestine crypt,overlapping with that of Brdu. Part II The effects and its mechanisms of FGFR3 in small intestine of mice after ischemia reperfusion injuryMain methods:1. Animals and groups: Mice with Fgfr3369/+ (Mutants) and their littermate controls (C57BL/6J) (controls).Six to eight weeks old mice were divided into normal control group,wild I/R group and FGFR3+/- I/R group,each group has 6 mice.Absolute diet 12 hours before operation,drinking water freely.Take anesthetization by 0.6% pentobarbital sodium intraperitoneally.Superior mesenteric artery(SMA) was occluded to produce ischemia of the intestine for 1 hour followed by reperfusion to reproduce I/R injury.The mice of normal control group were killed directly while the other two groups were killed after loosening arterial clamp 1hour,3hours,6 hours and 1day,3 days. Blood were collected from orbital vein sinus, then it was centrifuged at 3000 rpm for 10 minutes and then immediately stored in a -20°C icebox to determine plasma D-lactic acid level by Thermo Labsystems Multiskan Spectrum.About 1.5 cm long proximal jejulum was fixed,embedded and sliced routely,which was prepared to examine PCNA and apoptosis.The left bowel was immediately stored at -80°C to examine expression of FGFR3 mRNA and activity of ERK1/2.Main results:1. Progressively severe mucosal injury occurred 1,3,6h after reperfusion of the ischemic intestine.At the 1,3d group,the recovery of mucosal injury were observed, The damage in small intestinal epithelium were significantly lower in fgfr3+ I/R group than wild I/R group.2.Dramatically increased epithelial apoptosis occurred 1,3,6h after reperfusion of the ischemic intestine.At the 1,3d group, attenuation of epithelial apoptosis were observed.The apoptosis in small intestinal epithelium were significantly lower in fgfr3+ I/R group than wild I/R group.3.Dramatically increased crypt epithelial proliferation occurred 1,3,6h after reperfusion of the ischemic intestine.At the 1,3d group, proliferation activity gradually decreased.The proliferation were significantly higher in fgfr3+ I/R group than wild I/R group.4. The D-lactic acid level of both two groups were remarkably higher than that of normal group at every time point,and they both reached peak value at 1h after reperfusion,then gradually decreased,but the level was significantly lower in fgfr3+ I/R group than wild I/R group at 1,3,6h after reperfusion.5. The mRNA expression level of FGFR3 in small intestine of both two groups were remarkably elevated and they both reached the first peak at 1h after reperfusion,then quickly decreased to the ebb at 3h after reperfusion.They again significantly rose to the second peak and then gradually decreased,while they remained at a high level at 3d.6. The Erk level of both two groups were at constant level,while the p-Erk level of both two groups were remarkably higher than that of normal group after injury,and then gradually decreased,the mutant decreased slowly and the wild decreased to the normal level at 1d.Major conclusions:1. FGFR3 improves formation of murine intestine crypt in development phase.2. FGFR3 prohibited the apoptosis of intestinal epithelium and improved the proliferation after intestinal ischemia-reperfusion injury.3. FGFR3 can promote small intestinal epithelial recovery,which might be acquired through activation of ERK1/2 in small intestinal epithelium.更多还原