【作者】 张旋；

【导师】 姜小鹰；

【作者基本信息】 福建医科大学 ， 老年医学， 2012， 博士

【摘要】 目的：（1）研究脑外伤大鼠肠黏膜多巴胺受体表达的变化，探讨肠道多巴胺受体在脑外伤大鼠肠黏膜屏障功能损伤中的作用；（2）比较添加不同营养素的肠内营养支持方式对脑外伤大鼠肠黏膜多巴胺受体表达的影响及与肠黏膜屏障功能的关系，为临床危重症患者营养支持提供客观的理论依据。方法：采用改进后的Feeney自由落体脑损伤装置，造成大鼠中度脑损伤建立脑外伤大鼠模型。首先将大鼠随机分为正常对照组、脑外伤组、多巴胺组、拮抗剂组，分别于脑外伤第3、7天取肠黏膜组织、尿液、脏器组织等标本。通过测量大鼠的体重，用尿中乳果糖和甘露醇排泄率比值(L/M)测定肠黏膜的通透性，用HE染色法观察大鼠肠黏膜组织形态学的改变，用脏器组织中大肠杆菌菌落数测定细菌易位情况，用免疫组化方法检测肠黏膜多巴胺受体的表达，用荧光定量PCR和western－blotting法分别检测大鼠肠黏膜多巴胺受体表达的mRNA和蛋白水平，以此观察脑外伤大鼠肠黏膜屏障功能和多巴胺受体表达的变化。在前期研究基础上，将脑外伤大鼠随机分为对照组、肠内营养组、谷氨酰胺组、益生菌组、联合组，给予营养支持。分别于脑外伤第3、7天留取标本，采用上述方法观察添加不同营养素的肠内营养对脑外伤大鼠肠黏膜屏障功能和多巴胺受体表达的影响。收集各组资料，主要采用均数±标准差、单因素方差分析、LSD-t检验、SNK-q检验等进行统计学分析。结果：1.脑外伤大鼠肠黏膜屏障功能受损：（1）不同时间点各组大鼠尿中L/M比值存在差异（F=67.155，P<0.01；F=564.713，P<0.01），脑外伤组、多巴胺组和拮抗剂组大鼠的L/M比值均高于对照组（P<0.01）；与脑外伤组相比，多巴胺组与之比较无差异（P>0.05），而拮抗剂组则较之有所降低（P<0.01）；与第3天比较，第7天对照组（t=1.152）、脑外伤组（t=0.878）和多巴胺组（t=0.780）的L/M比值无差异（P>0.05），拮抗剂组比值则下降（t=2.84，P<0.05）。（2）HE染色结果显示：脑外伤组和多巴胺组大鼠在第3天，肠黏膜绒毛完整性被破坏，绒毛间质疏松、肠绒毛排列极不规则，绒毛上皮坏死脱落，上皮层与固有层可见分离；第7天，肠黏膜上皮出现变性萎缩，绒毛变矮，间距增宽。但拮抗剂组大鼠绒毛变化较脑外伤组和多巴胺组程度为轻，只有轻度水肿。（3）脑外伤组大鼠肠、肺、肝、脾、肾等脏器中均检测出大肠杆菌，各组大鼠在肠、肝、脾、肾等脏器中的大肠杆菌的菌落数比较组间有差异（P<0.01）。2．脑外伤大鼠肠黏膜多巴胺受体表达水平上调：（1）脑外伤组大鼠肠黏膜上皮结构破坏，可见大量的棕褐色阳性反应产物，随着时间的增加，棕褐色阳性反应产物也有所增加；多巴胺组大鼠肠黏膜上皮结构基本完整，但可见大量的棕褐色阳性反应产物；拮抗剂组大鼠第3天肠黏膜上皮细胞可见大量的棕褐色阳性反应产物，但第7天棕褐色阳性反应产物有所减少。（2）各组大鼠肠黏膜DRD1和DRD2表达的mRNA水平的PCR检测发现，与对照组相比，脑外伤组、多巴胺组和拮抗剂组的DRD1和DRD2表达的mRNA水平均升高（P<0.01），与脑外伤组相比，多巴胺组受体的表达mRNA水平无差异，拮抗剂组则较前两组有所降低。WB检测结果显示，各组受体表达均较对照组上调，其中，脑外伤组和多巴胺组表达DRD1和DRD2的条带亮度无明显差别，拮抗剂组条带亮度则有差异。3．添加不同营养素的肠内营养方式有助于改善脑外伤大鼠肠黏膜屏障功能：（1）脑外伤后各组大鼠体重均有不同程度的降低（P<0.05），但各组间大鼠体重存在差异（F=6.30，P<0.01），与对照组比较，添加不同营养素的肠内营养组大鼠的体重均有所增加（P<0.01）。（2）脑外伤第7天，各组大鼠肠黏膜L/M比值存在差异（F=432.472，P<0.01），添加了不同营养素的肠内营养组大鼠肠黏膜L/M比值均较对照组有所降低，其中益生菌组大鼠L/M比值要低于其他营养组（P<0.05）。（3）HE染色结果显示，添加了不同营养素的各组大鼠较脑外伤组的病理损伤均有不同程度的减轻。（4）脑外伤第7天各组大鼠肠、肺、肝、脾、肾等脏器中均检测出大肠杆菌，在肠、肺、脾、肾等脏器，各组大肠杆菌数比较组间有差异，均少于对照组（P<0.05），益生菌组和联合组检测出在肠道的菌落数要少于肠内营养组和谷氨酰胺组（P<0.01）。4．添加不同营养素的肠内营养方式可使肠黏膜多巴胺受体表达水平下调：（1）脑外伤第7天，各营养组大鼠肠组织棕褐色阳性反应产物有所减少。（2）各组大鼠肠黏膜DRD1和DRD2表达的mRNA水平的PCR检测发现，与对照组相比，各营养组的DRD1和DRD2表达的mRNA水平均下降（P<0.01）；各营养组的DRD1表达mRNA水平无明显差异；益生菌组和联合组大鼠肠黏膜的DRD2表达mRNA水平较肠内营养组和谷氨酰胺组有所下降（P<0.01）。WB检测结果显示，各组受体表达均较对照组下调，表达DRD1和DRD2的条带亮度无明显差别。结论：（1）脑外伤可导致大鼠肠黏膜通透性明显升高，肠黏膜上皮细胞受损、细胞间紧密连接增宽等组织形态学的改变，以及发生脏器组织细菌易位等现象，表明脑外伤大鼠肠黏膜屏障功能受损。（2）脑外伤大鼠肠道多巴胺受体的表达水平增加，说明脑外伤大鼠肠黏膜屏障功能受损可能与肠道多巴胺受体表达水平的上调有关。（3）添加不同营养素的肠内营养能够减缓脑外伤大鼠体重的下降趋势，阻止脑外伤大鼠肠黏膜通透性的增加，减轻脑外伤大鼠肠黏膜组织形态的损伤，对脑外伤大鼠肠黏膜屏障具有保护作用，有助于减少肠道细菌易位的发生。添加不同营养素的肠内营养在减缓脑外伤大鼠体重下降趋势以及维护肠黏膜屏障功能方面各有优势，这可能与不同营养素对改善肠黏膜屏障功能的不同机制有关。（4）添加不同营养素的肠内营养能下调肠道多巴胺受体的表达水平，通过减少多巴胺与其受体的结合，从而对肠黏膜屏障功能起到保护作用，这可能是添加不同营养素的肠内营养制剂改善脑外伤大鼠肠黏膜屏障功能的机制之一，为危重症患者的临床营养支持与护理提供了理论基础。更多还原

【Abstract】 OBJECTIVE:The purposes of this study are (1) to research the expression changes of dopaminereceptor on intestinal mucosa of rats with traumatic brain injury, and explore the roleof intestinal dopamine receptors in the intestinal mucosal barrier function；(2) tocompare the effection of adding different nutrients to enteral nutrition on intestinalmucosa of rats with traumatic brain injury and the relationship between the expressionof dopamine receptor and intestinal mucosal barrier function, and provide objectivetheoretical basis for nutritional support on the clinical critically ill patients.DESIGN:Using the improved Feeney free-fall brain injury unit, resulting in rat moderate braininjury to establishing the traumatic brain injury rat model. The rats were randomlydivided into normal control group, the TBI group, DA group and dopamine antagonistgroup, respectively in3,7days after traumatic brain injury of intestinal mucosal tissue,urine, viscera and tissue specimen. By measuring the weight of rats, using urinarylactulose and mannitol excretion rate ratio（L/M）determination of intestinal mucosalpermeability, with HE staining of rat intestinal mucosa morphology changes, withinternal organs in Escherichia coli colony number determination of bacterialtranslocation, using immunohistochemical method for the detection of intestinalmucosa of dopamine receptor expression, by PCR and Western-blotting method wereused to detect the intestinal mucosa of rats with dopamine receptor expression onmRNA and protein level, as observed in traumatic brain injury rat intestinal mucosalbarrier function and dopamine receptor expression changes. In the early researchfoundation, the traumatic brain injury in rats were randomly divided into controlgroup, enteral nutrition group, glutamine group, the probiotic group, united group, nutrition support. In traumatic brain injury in third,7days of taking samples, themethod of adding different nutrients on enteral nutrition on traumatic brain injury inrat intestinal mucosa barrier function and dopamine receptor expression in the rat.Data was collected, mainly uses the mean+SD, single factor analysis of variance,LSD-t test, SNK-q test and statistical analysis.RESULTS:1．traumatic brain injury of intestinal mucosa in rats with impaired barrier function:(1)at different time points in each group rats the urinary L/M ratio difference (F=67.155, P <0.01; F=564.713, P <0.01), traumatic brain injury group, dopamine andantagonists in rats of the L/M ratio was higher than that of control group (P <0.01);and the traumatic brain injury group, dopamine group with no differences (P>0.05),while the antagonist group compared with the reduced (P <0.01); and the third days,seventh days of the control group (t=1.152), traumatic brain injury group (t=0.878)and dopamine group (t=0.780) and L/M ratio no difference (P>0.05), the ratio ofantagonists of group decreased (t=2.84, P <0.05).(2) the HE staining showed that:compared to the control group in the rat intestinal mucosa morphology is basicallynormal, intestinal mucosal villous structure integrity, villous epithelial cells arrangedin rows, arranged in rows, interstitial edema and free; traumatic brain injury groupand dopamine in rats during traumatic brain injury for third days, intestinal villiintegrity is destroyed, intervillous interstitial hyperemia and edema, interstitialosteoporosis, intestinal villi are extremely irregular, villus epithelial necrosis andexfoliation, epithelium and lamina propria visible separation, lamina propria appearedvascular dilation, congestion and edema; seventh days after injury, intestinal mucosalepithelium appeared atrophic villi, shorter, wider spacing, interstitial still hyperemiaedema. But the antagonist rats villous changes more traumatic brain injury group anddopamine group for light, with only mild edema.(3) in traumatic brain injury rats gut,lung, liver, spleen, kidney and other organs are detected in the Escherichia colibacteria of the genus, the rats in the intestine, liver, spleen, kidney and other organs inEscherichia coli bacteria of the genus colony number compare differences (P <0.01).Compared with the control group, the TBI group and dopamine in rats in the intestine, liver, kidney and other organs in bacterial colony number (P <0.05), traumatic braininjury group and dopamine in rats in the gut, liver and other organs of bacterial colonycounts difference (P <0.05), while the antagonist group and control group nodifference (P>0.05).2. traumatic brain injury of intestinal mucosa in rats with dopamine receptorexpression levels up:(1) was observed under microscope control group DRD1isuniformly continuous distribution in intestinal epithelial cell edge, top along the villuscell membrane, under continuous distribution; traumatic brain injury rat intestinalepithelial structure destruction, there are a lot of Tan positive reaction, with the timeincreased, Tan positive reaction also increased; dopamine rats intestinal epithelialstructure largely intact, but there are a lot of Tan positive reaction; antagonist groupthird days in rat intestinal epithelial cells visible Large Tan positive reaction, butseventh days Brown positive reaction decreased.(2) groups of intestinal mucosa ofrats with DRD1and DRD2expression in mRNA level of PCR testing found that,compared with the control group, the TBI group, dopamine and antagonists of groupDRD1and DRD2expression of mRNA water was increased (P <0.01), and traumaticbrain injury group, dopamine receptor expression level of mRNA group nodifferences, antagonist group than the other two groups decreased. WB detects theresult shows, each receptor expression than those in the control group raised, wherein,traumatic brain injury and expression of DRD1and DRD2dopamine group bandbrightness difference, antagonists of group band brightness are different.3. add different nutrition enteral nutrition can help improve brain trauma in the ratintestinal mucosal barrier function:(1) after traumatic brain injury in rats body weightwere significantly reduced (P <0.05), but the group rats body weight differences (F=6.30, P <0.01), and the control group comparison, adding different nutrition enteralnutrition group rats body weight was increased (P <0.01), no differences between thenutrition group.(2) Traumatic brain injury seventh days, groups of intestinal mucosaof rats with L/M ratio difference (F=432.472, P <0.01), add different nutritionenteral nutrition group of intestinal mucosa in rats with L/M ratio was lower than thecontrol group, the difference was statistically significant (P <0.01). One of the probiotic group rat L/M ratio is lower than the other nutrition group (P <0.05).(3)the HE staining showed that, add different nutrients in rats with traumatic brain injurygroup of pathological damage in varying degrees of loss.(4) of traumatic brain injuryon day seventh rats in each group, lung, liver, spleen, intestine, kidney and otherorgans are detected in the Escherichia coli bacteria of the genus, in the gut, lung,spleen, kidney and other organs, groups of Escherichia coli bacteria of the genuscolony number compare differences, were lower than the control group (P <0.05),the probiotic group and combination group detected in the intestinal colony numberless than enteral nutrition group and glutamine group (P <0.01).4. add different nutrition enteral nutrition mode can make the intestinal mucosa ofdopamine receptor expression levels by:(1) after traumatic brain injury in ratsintestinal tissue were visible Brown positive reaction. Compared with the controlgroup, brain trauma seventh days, the nutrition group of rat intestinal tissue Brownpositive reaction decreased.(2) groups of intestinal mucosa of rats with DRD1andDRD2expression in mRNA level of PCR testing found that, compared with thecontrol group, the nutrition group DRD1and DRD2expression of mRNA water dropson average (P <0.01); the nutrition group DRD1expression level of mRNA had nosignificant difference; the probiotic group and combination group of rat intestinalmucosal DRD2expression of mRNA levels than the enteral nutrition group andglutamine group decreased (P <0.01). WB detects the result shows, each receptordownregulation of expression than those in the control group, DRD1and DRD2bandbrightness difference.CONCLUSION:(1) traumatic brain injury can lead to intestinal mucosa permeability in rats wassignificantly elevated, the epithelial cells of intestinal mucosa injury, intercellulartight junctions wider tissue morphological changes, and the occurrence of organ tissuebacterial translocation and other phenomena, suggests that traumatic brain injury ofintestinal mucosa in rats with impaired barrier function.(2) in traumatic brain injury rat intestinal dopamine receptor expression levelsincreased, that traumatic brain injury of intestinal mucosa in rats with impaired barrier function may be associated with intestinal dopamine receptor expression levels relatedto the up-regulation of.(3) adding different nutrition enteral nutrition can slow brain trauma in the rat bodyweight decreased, prevent traumatic brain injury in the rat intestinal mucosapermeability increase, reduce brain injury in the rat intestinal mucosa tissuemorphology of injury, on intestinal mucosal barrier of rats with traumatic brain injuryhas a protective effect, helps to reduce bacterial translocation the occurrence of.Adding different nutrition enteral nutrition in slow brain trauma in the rat weightdecreased and the maintenance of gut barrier function have their respectiveadvantages, which may be associated with different nutrients on intestinal mucosalbarrier function by different mechanisms related to.(4) adding different nutrition enteral nutrition can down-regulate intestinal dopaminereceptor expression levels, through the reduction of dopamine and its receptor binding,thereby on intestinal mucosal barrier function plays a role in protection, it may beadded with different nutrition enteral nutrition improvement in traumatic brain injuryrat intestinal mucosal barrier function is one of the mechanisms, critically ill patientswith clinical nutritional support and nursing care provided academic basis.更多还原