摘要
多器官功能障碍综合征(multiple organ dysfunction syndrome，MODS)是创伤及感染后最严重的并发症，发病率高，往往由于来势凶猛，病情发展急剧，难以被迄今的器官支持治疗所遏制，死亡率很高。肠道被认为是MODS的启动器官，许多研究证实，严重创伤和感染早期，肠黏膜屏障功能减退、肠道微生态失衡以及肠黏膜免疫功能失调，大量炎症介质如TNF-α、IL-1等产生，引起全身炎症反应综合征(system inflammatory reaction syndrome，SIRS)，导致器官功能障碍，最终发展为MODS。
肠黏膜免疫系统是一个完整的组织网络，由Peyer淋巴结、肠黏膜固有层淋巴细胞、上皮间淋巴细胞(intraepithelial lymphocytes，IELs)组成。其主要的效应细胞有CD4~+T细胞、CD8~+T细胞、B细胞、分化的浆细胞、巨噬细胞和抗原呈递细胞等；主要的效应分子是SIgA。黏膜免疫活动包括肠淋巴细胞活化、归巢和SIgA分泌等。它的主要功能是参与机体的免疫防御，如参与机体的过敏反应、超敏反应和炎症。近年来，越来越多的研究表明，肠黏膜损伤在MODS发生发展中起着重要作用，但构成肠黏膜屏障的重要组成部分——肠黏膜免疫系统在MODS中的作用却不清楚。
肠淋巴细胞归巢作为肠黏膜免疫系统重要免疫活动之一，它参与了肠黏膜急、慢性炎症的发生；使肠黏膜有效地行使免疫监视和免疫清除功能；是肠黏膜免疫系统与系统免疫系统沟通的重要桥梁。胃肠多肽、细胞黏附分子及一些细胞因子可能对淋巴细胞的迁移产生重要影响。然而MODS时肠黏膜淋巴细胞迁移的规律及其调控机制却并不清楚。
此外，肠黏膜的神经细胞与神经介质、内分泌细胞与多肽分子、免疫细胞与细胞因子都是机体中数量最集中的部位。胃肠多肽是神经-内分泌-免疫网络中的重要调节因子。研究表明，肠黏膜固有层和Peyer淋巴结密集分布着肠血管活性多肽(vasoactive intestinal polypeptide，VIP)和生长抑素(somatostatin，SST)等肽能神经纤维，这些肽能神经纤维和上皮层及固有层的淋巴细胞，巨噬细胞等免疫细胞毗邻，直接调节着肠黏膜免疫系统的功能。此外，特异性VIP受体广泛存在于Peyer淋巴
重庆医科大学博士学位论文
结高内皮小静脉(high endothelial venule，HEV)及淋巴管内部和周围，以及滤泡边缘
的T细胞中，SST受体也在小鼠Peyer淋巴结和人肠豁膜固有层淋巴细胞中被发现，
这些受体可能对于淋巴细胞通过Peyer淋巴结或肠豁膜固有层的迁移具有重要的
调节作用。但VIP或SST对肠淋巴细胞迁移调节的研究不多，至于它们对MODS时
肠淋巴细胞调节的研究尚未见报道。
研究目的
l、探讨生理情况下:
1) VIP或SST对大鼠肠淋巴细胞向系统免疫系统迁移的影响;
2) vIP或ssT对大鼠肠淋巴细胞归巢至肠相关淋巴组织(gut一associated
lymphoid tissue，GA叮)的调节;
2、MODS时肠淋巴细胞再循环的改变，从豁膜免疫的角度探讨MODS的病理生
理机制:
3、VIP或SST对MODS转归的影响;
4、MODS时VIP或SST对肠淋巴细胞归巢的调节，从豁膜免疫的角度理解MODS
的病理生理机制;
5、viP或SST是否通过肠淋巴细胞上的相应受体调节肠淋巴细胞的归巢?
6、viP或SST对存在于小肠内皮细胞上决定肠淋巴细胞归巢到GALT的关键豁附
分子一豁膜地址素细胞豁附分子一1(mucosal“枷ssin eella曲esion moleeule一l，
MAdCAM一l)的影响，以确定这两种胃肠多肤调节肠淋巴细胞归巢的分子机制。
研究方法
1、从大鼠肠系膜淋巴管插管引流淋巴液，收集肠淋巴细胞;
2、E花结分离法测定T细胞比例;
3、流式细胞仪检测肠淋巴CD4+、CDS+淋巴细胞;
4、slCr标记肠淋巴细胞示踪其在GALT的分布;
5、采用微型无创动脉夹夹闭肠系膜上动脉造成肠缺血再灌注损伤，诱导大鼠
MODS模型;
6、尸F冈F一a ELISA试剂盒测定肠淋巴液和外周血TNF一a水平;
重庆医科大学博士学位论文
7、酶学分光光度法测定外周血D一乳酸水平;
8、赏试剂盒测定肠淋巴液和外周血内毒素水平;
9、免疫组织化学法检测肠淋巴细胞T、B细胞比例;
10、苏木素一伊红染色法观察大鼠小肠、肝脏、肾脏和肺脏的组织学改变;
11、采用Rl斗PCR方法检测肠淋巴细胞上SST和VIP受体的mRNA表达;
12、采用W七stem blot方法检测小肠组织MAdCAM一1表达。
研究结果
1、生理情况下，由肠勃膜进入血循环的肠淋巴量、单位体积淋巴细胞数及单
位时间淋巴细胞数保持稳定;由血循环迁移至GALT的肠淋巴细胞比例:Peyer
淋巴结为3.83%士1.87%，小肠弥散淋巴组织为1.07%士0.61%。
2、viP(o.2Pmol.g一’·h一‘、7 hour)显著降低肠淋巴细胞由肠勃膜向血循环的迁移:
持续从静脉输入vIP后，5小时肠淋巴液中淋巴细胞总数为6.92士0.63、107个，显
著低于对照组( 9.43士1.38/107个)，P<0.05，降低26石%，而肠淋巴液量无显著
改变，尸>0.05，其中，T细胞降低20.6%，CDS+细胞降低1 1 .91%，CD4+细胞无显
著改变;同时，vIP还抑制肠淋巴细胞由血循环进入GALT:Peyer淋巴结’‘cr-肠
淋巴细胞量系体内总”c卜肠淋巴细胞量的1.83%士0.90%，显著低于对照组3.85%
Multiple organ dysfunction syndrome (MODS) is the most serious result induced by trauma and infection. Once established, MODS defies our supportive measures. Mortality ranges from 40% to 100% and is related directly to the number and duration of organ failures. Unfortunately, neither the incidence nor the mortality of the syndrome has improved significantly. MODS is regarded as a continuous inflammatory response that is unable to be controlled. Intestinal mucosal barrier function disturbance, including intestinal mucosal immune dysfunction, play a vital role during the development of the systemic inflammatory response syndrome which causes important organs or tissues injury and then runs to MODS.
The mucosal immune system consists of an integrated network of tissues, including the lymphoid cells in Peyer's pathes and lamina propria, and intraepithelial lymphocytes. Major effector cells include CD4+ T cells, CD8+ T cells, B cells, differentiated plasma cells, macrophages, and antigen-presenting cells (APCs) and so on. Major effector molecules include antibodies, largely of the immunoglobulin A (IgA) isotype as well as cytokines, chemokines, and their receptors. Lymphocytes activated, lymphoid cell homing, and SIgA secrete are taken as the main mucosal immune response.
Accumulative studies show that the injury of intestinal mucosa plays a vital role at the beginning and development of MODS. However, the role of intestinal mucosal immune system, as an important part of intestinal mucosa barrier, especially in the case of MODS remains still unclear.
Intestinal lymphocyte homing, as one of an important immune activites, conducts the function of immune surveillance and immune cleaning and therefore is involved in the acute and chronic inflammation of intestinal mucosa. It is an important trafficking bridge between intestinal mucosal immune system and systemic immune system. Gut peptides, cell adhesion molecules, and other cytokines have shown an important effect on the migration of lymphocytes. However, the knowledge on the regulation of intestinal mucosal lymphocyte homing in the situation of normal or MODS is still limited.
In addition, intestinal mucosa is the place plenty of neurocytes and transmitters, endocrinocyte and polypeptides, immunologically competent cells and cytokines. Gut peptides are important regulators in the neuro-endocrine-immune network. VIP, SST and other peptidergic nerve fibers, which are near various immune cells, are found in intestinal laminal propria and Peyer's patches with high concentration or density. They are capable of regulating the lymphocyte migration here. However, the effects of gut peptides on intestinal lymphocyte homing, especially in the case of MODS remain unclear.
AIMS
To investigate:
1. The migration behavior of intestinal lymphocyte at gut-associated lymphoid tissue (GALT) in the physical state;
2. The regulation of VIP or SST on homing of intestinal lymphocyte at GALT in normal rats;
3. The changes of homing of intestianl lymphocyte at GALT in rats with MODS;
4. The effects of VIP or SST on homing of intestinal lymphocyte of rats with MODS;
5. Whether intestinal circulating lymphocyte expresses mRNA of receptors for SST and VIP;
6. The effect of VIP and SST on expression of MAdCAM-1, which exists in high endothelial venules of Peyer's patches and intestinal lamina propria and targets migration of intestinal lymphocyte.
METHODS
1. Intestinal lymphocyte was collected from rat intestinal lymphatics by a plastic tube.
2. The percentage of T lymphocytes in intestine lymph liquid was determined by E rose ring.
3. The percentage of CD4+ and CD8+ T lymphocytes in intestine lymph liquid was measured by flow cytometry.
4. 51Cr-labeled lymphocyte distributed in GALT was counted with -counter.
5. The rat model of MODS was established by intestine isochemia-reperfusion.
6. Tumor necrosis factor a (TNF- ) in plasma and intestinal mucosa was determined with a ELISA Kit for TNF- .
7. D(-)-Lactate level in plasma was m
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