【作者】 刘苓；

【导师】 唐承薇；

【作者基本信息】 四川大学 ， 内科学， 2007， 博士

【摘要】 研究背景和目的：肠缺血再灌注（intestinal ischemia-reperfusion，IIR）是发生多器官功能障碍综合征（multiple organ dysfunction syndrome，MODS）的重要病理生理过程。全身炎症反应（systemic inflammatory response syndrome，SIRS）是IIR后MODS发生的重要发病机理，早期干预炎症反应，积极防治MODS，是降低危重病患者病死率的重要手段。全身系统性炎症反应的发生取决于机体的免疫系统。传统上免疫系统分为天然免疫和获得性免疫。我们课题组前期工作表明猕猴IIR后并发SIRS及MODS的发病过程中，肠粘膜TLRs-NF-κB-炎症介质信号转导通路全面激活，但IgA、SC、SIgA均显著减少，表现为肠粘膜过强天然免疫与降低的获得性体液免疫共同存在。IFN-γ是免疫炎症反应的一种重要调控因子，对天然免疫和获得性免疫均有调控作用，但IFN-γ是否参与IIR后肠粘膜免疫反应及SIRS的形成?如果参与，是通过何种机制?细胞因子常与其它激素，神经肽、神经递质共同组成细胞间信号分子系统，如果IFN-γ参与了肠黏膜免疫活动，生长抑素（somatostatin，SST）是否影响IFN-γ的产生?通过这方面的研究，以期增进我们对IIR后肠黏膜免疫的认识，并为防治SIRS、MODS提供新的思路。急性重症胰腺炎（severe acute pancreatitis，SAP）是导致IIR的常见病因，在SAP—IIR—MODS的发生中，IIR反过来对胰腺组织病理及功能又有何影响?自SST用于治疗SAP以来，有效降低了MODS发病率。SST的这些作用究竟仅仅是因为抑制了胰酶分泌，还是可以避免IIR后SIRS对胰腺的再次打击?这方面的研究将更新临床防治MODS或SAP的思路，并增加我们对SST在抗炎药理方面的认识。传统认为，B细胞介导的获得性免疫在肠粘膜免疫中具有重要的作用。我们研究小组前期的实验表明IIR后，大鼠肠淋巴细胞归巢增加，猕猴PP（peyer’s patches，PP）内B细胞明显增多，但是肠粘膜获得性体液免疫效应却表现为sIgA减少。新近研究表明，B淋巴细胞具有获得性免疫及天然免疫的双重功能。那么IIR时PP内大量集聚的B细胞是否参与肠黏膜增强天然免疫的形成?如果有，是哪一类或哪一发育阶段的B细胞?参与天然免疫的B细胞是否包括具有免疫记忆的B淋巴母细胞?这些研究能增进我们对临床防治SIRS及MODS分子靶点的新认识。方法：1．实验动物：全部实验所用健康成年猕猴来自成都野生动物世界，雌雄不限，体重6.9±1.7Kg。猕猴进出成都野生动物世界猕猴喂养区时均检疫合格。2．实验分组：15只健康成年猕猴随机分为正常、IIR、IIR+SST三组，每组5只动物。肠缺血再灌注手术：肌注复方二甲苯胺噻嗪（0.06～0.24ml／kg）麻醉动物，实验过程中用安定静注0.16±0.09mg／kg／h维持昏睡状态。无菌铺巾，沿腹正中切开，分离肠系膜上动脉后予以夹闭，60 min后松夹进行再灌注，恢复肠系膜上动脉血流。再灌注后1h，静脉输入0.9％盐水，0.1～0.2ml／kg／min，24h内补入葡萄糖20g。3．预防使用SST：在夹闭肠系膜上动脉前，从静脉滴注生长抑素，5μg／kg／h，持续至再灌注后24h。4．HE染色法观察回肠、胰腺组织病理损伤程度。5．放射免疫分析检测外周血SST的含量。6．ELISA检测肠上皮细胞、肠淋巴细胞、肠淋巴细胞孵育上清液、回肠组织匀浆、外周血清、门静脉血清中IFN-γ的水平。7．ELISA检测外周血清及回肠组织匀浆中IL-1β、IL-6、TNF-α水平。8．酶比色法检测血清淀粉酶及脂肪酶。9．免疫组化检测IFN-γ、CD4、CD8、CD57、CD20、CD5、PAX-5、浆细胞、TLR4、TLR2、NF-κB、integrinα4β7的分布，图像分析软件测定阳性面积及IOD值。10．门静脉血培养。11．培养人B淋巴母细胞株hmy2.cir，细胞干预组加入LPS（10μg／ml），另一组为无干预的空白对照组，37℃，5％CO₂孵育。12．ELISA检测干预24小时后培养上清液中IL—6及干预72小时后培养上清液中IgM的水平。13．免疫荧光检测hmy2.cir细胞TLR2、TLR4表达。14．MTT检测LPS干预hmy2.cir细胞株后24小时、48小时、72小时的增殖情况结果：1．IIR后回肠粘膜上皮细胞、门静脉血内IFN-γ水平较正常组显著增加（分别为30.7±20.1 pg／mg vs 9.4±2.4pg／mg总蛋白；18.1±1.1pg／ml vs 8.7±1.3 pg／m1），p＜0.05，而肠淋巴细胞及其上清液的IFN-γ水平较正常组明显降低（分别为1.7±0.02pg／mg vs 11.1±4.0pg／mg总蛋白；2.2±0.3pg／mg ys 5.2±1.5pg／mg）（p＜0.05）；2．预防性给予SST后，与IIR组比较，回肠上皮细胞及门静脉血内IFN-γ的水平显著下降（分别为13.6±4.9pg／mg总蛋白，p=0.055；8.9±2.5pg／ml，p＜0.05），肠淋巴细胞及其上清液的IFN-γ水平无明显变化；3．组织匀浆及外周血的IFN-γ水平在三组间无明显差别。CD4⁺细胞、CD57⁺细胞分布与IFN-γ无关，CD8⁺细胞分布与IFN-γ变化趋势相反。4．IIR后小肠黏膜PP数量增加、增大。5．正常组猕猴肠黏膜PP内的B细胞均为PAX-5⁺ CD20⁺ CD5^-，系B-2细胞，黏膜固有层有散在浆细胞。IIR后PP内PAX-5⁺细胞显著增加（p＜0.05），但其中CD20表达未见增多，CD5^-表达，出现PAX-5⁺ CD20^- CD5^-的祖B细胞，黏膜固有层内浆细胞显著减少（p＜0.05）。6．正常时，PP中TLR2、TLR4、NF-κB及IFN-γ表达均较弱，IIR时PP内四种分子表达均明显增强。7．正常PP内有散在α4β7表达，IIR组明显减少。8．IIR后，猕猴外周血及回肠黏膜组织中炎症介质IL-1β（外周血：79.2±14.4ng／ml；回肠：294.0±46.4pg／g）、IL-6（外周血：1261±297.5ng／ml；回肠：1527±160.8p／g）、TNF-α（外周血：64.8±18.7ng／ml；回肠：213.2±29.2pg／g），与正常组IL-1β（外周血：27.3±7.17ng／ml；回肠：82.8±20.5pg／g）、IL-6（外周血：13.9±10.50ng／ml；回肠：709.6±211.2pg／g）、TNF-α（外周血：3.04±1.01ng／ml；回肠：56.0±10.04pg／g）比较，均显著增加（p＜0.05）。SST治疗后，外周血中IL-1β（40.0±9.9ng／ml）、IL-6（244.4±70.0 ng／ml）、TNF-α（19.2±10.1 ng／ml）水平明显下降，与IIR组相比有显著性差异（p＜0.05）。9．正常组5只动物的门静脉血培养均为阴性；IIR组所有猕猴血培养均有细菌生长，阳性率为100％，而且引起菌血症的细菌均为大肠杆菌。10．LPS干预72小时后，hmy2.cir细胞上清液中IgM水平为0.528±0.002 ng／ug总蛋白，与空白对照组（0.617±0.057ng／ug总蛋白）相比显著减少（p＜0.05）。11．LPS干预72小时后，与对照组相比，hmy2.cir细胞TLR4的IOD（1201.3±913.4 vs 5078.2±2483.2）及TLR2的IOD（174.8±80.2 vs 1463.5±51.2）较对照组显著减少（p＜0.05）。12．LPS干预24小时后，hmy2.cir细胞上清液中IL-6水平（0.528±0.002 pg／ug总蛋白）与空白对照组（0.406±0.218pg／ug总蛋白）相比显著减少（p＜0.05）。13．LPS干预24小时后，hmy2.cir细胞增殖水平（0.389±0.061）较空白对照组（0.727±0.245）为低，72小时后细胞增殖水平（0.562±0.219）较对照（0.173±0.079）显著增加（p＜0.05）。14．IIR后，所测胰淀粉酶（2 492.0±2 102.8 IU／L vs 385.4±136.1 IU／L）及脂肪酶（1 278.0±1 446.3 IU／L VS 25.8±13.0 IU／L）在血中水平较正常组均显著升高（p＜0.05）。预防性使用SST，既使发生IIR，血胰淀粉酶（567.0±338.0 IU／L）及血脂肪酶（77.2±779 IU／L）明显回落。15．放射免疫分析结果表明，IIR后外周血SST（62.17±13.56 pg／ml）较正常对照（160.61±33.84 pg／ml）明显下降（p＜0.05），静脉补充SST后，循环SST水平（156.32±30.25 pg／ml）显著回升（p＜0.05）。16．IIR时可见胰腺组织病理损伤。预防性使用SST后，胰腺病理组织学较IIR时明显减轻，组织学评分从+++降到++。结论：1．IIR后，来源于上皮细胞及早期祖B细胞大量产生IFN-γ，后果是通过肠、黏膜、肺泡肥大细胞上的FcεRIα活化肥大细胞，引起强烈的天然免疫反应。换言之，IFN-γ将TLR-NF-κB与肥大细胞两个天然免疫反应通路联系在一起，构成了复杂、高效的天然免疫网络。2．IIR后祖B细胞参与肠黏膜天然免疫反应，在肠黏膜屏障严重损伤时常伴发LPS血症，这使得LPS可在循环甚至骨髓中通过祖B细胞的TLR介导使祖B细胞迅速卷入天然免疫，在循环中产生大量IL-1β、IL-6、TNF-α，这应是SIRS发生的重要原因之一。3．IIR后肠黏膜PP内PAX-5⁺ CD20⁺ CD5^-、具有免疫记忆的B淋巴母细胞并未在强烈、快速反应的天然免疫中发挥明显作用。4．整个B淋巴细胞群具有天然免疫及获得性免疫的双重功能。在不同发育阶段的B淋巴细胞仅分别发挥其中某一项功能。代表早期发育阶段的祖B淋巴细胞高表达TLRs，且能被LPS激活，经TLR-NF-κB-促炎因子途径发挥快速、强烈的天然免疫功能；成熟B细胞具有较弱的天然免疫作用，主要参与获得性免疫；代表发育晚期阶段的B淋巴母细胞虽有TLRs表达，但不能被LPS激活，不具有天然免疫功能。5．IIR导致MODS时，胰腺应为值得重视的、早期受损的器官。其炎症组织病理改变，不是缺血后氧化应激所致，而与循环中炎症介质进入胰腺后激活NF-κB有关。SST可通过降低全身炎症反应，解除血炎症介质对胰腺的再次损伤，从而避免急性胰腺炎向重症方向发展。更多还原

【Abstract】 BackgroundIntestinal ischemia-reperfusion （IIR） is an important pathophysiological factor, contributory to systemic inflammatory response syndrome （SIRS）, resulting in multiple organ dysfunction syndrome （MODS）. Early intervention on the inflammatory response during IIR is an effective way to reduing MODS-associated mortality of critically ill patients.Development of SIRS depends on body’s immune response that consists of adaptive immunity and innate immunity. Our prior study revealed activation of TLRs-NF-[kappa]B-proinflammatory gene expression, but decreased IgA, SC, SIgA in intestine during IIR macaque, reflecting excessive innate immunity and reduced adaptive immunity in intestinal mucosal immunity. IFN-γis an important cytokine responsible for regulation of adaptive and innate immunity. However, whether and how IFN-γ, is involved in intestinal mucosal immunity and SIRS post IIR remains to be determined. Cytokines always works closely with other hormones, neuropeptides and neurotransmitters, constituting a particular intercellular network. Role of somatostatin （SST）, as a kind of important physiological neuropeptide in the production and regulation of IFN-γis also needed to characterize. Our research may help with understanding intestinal mucosal immunity after IIR and providing a new insight into treatment of SIRS and MODS.Intestinal ischemia reperfusion plays an important role in pathophysiological process of sever acute pancreatitis （SAP） to multiple organ disfunction. It is unclear if IIR aggravate inversely the inflammatory and histopathologic changes of pancreas. With the use of SST in treating SAP patients, it remarkedly reduced the incidence of MODS after SAP. Is it the result of suppressing the production of serum amylase levels? Does SST have a pivotal role in preventing second hit of pancreas during SIRS secondary to IIR? By answering this question, it will change our thoughts on prevention and treatment of SIRS and MODS, and will help to understand pharmacological mechanism of anti-inflammatory of SST.Traditionally, adaptive immunity mediated by B lymphocyte is regarded as an important factor in intestinal mucosal immunity. Our prior study indicates that increased homing of intestinal mucosal lymphocytes is involved in intestinal ischemia-reperfusion of rats; B lymphocytes in macaques peyer’s patches are obviously increased after IIR. But sIgA, as a feature of intestinal acquired immunity, is found to decrease. Recent studies have proved that that B lymphocytes is involved in innate immunity and in acquired immunity. Do B lymphocytes aggragating in peyer’s patches participate in excessive intestinal innate immunity? If yes, which type or which stage B lymphocytes have such function? Do immunological memory B lymphoblasts participate in innate immunity? By answering these questions, we should recognize new checkpoints and are able to understand the mechanism of SIRS and MODS better.Material and Methods1. 15 macaques with weighing 6.9±1.7Kg obtained from ChengDu safari park （ChengDu, china） were divided into three groups: normal control, IIR group and IIR+SST group.2. The animals were anesthetized with intramuscular xylazine compound （The military veterinary institute, quartermaster university of PLA, Changchun, China） at a dosage of 0.2±0.1ml/kg, followed by intravenous administration of diazepam at a dose of 0.16±0.09mg/kg/h. In IIR and IIR+SST group, A 5-cm vertical median incision at the epigastrium was made in animals after asepsis and antisepsis were completed. About 20% of initial blood volume removed [removed blood volume（ml）=weight（g）×5%] from inferior caval vein with a syringe and partial blood samples were used for organ functional laboratory tests. The superior mesenteric artery （SMA） was isolated near its aortic origin and was looped with a 7 silk suture. The SMA was occluded for 1h and reopened with this silk suture loop and a vascular clamp. During the experiment, the abdominal wall was kept approximated to prevent fluid and heat loss. One hour later, normal saline solution containing 20g glucose was infused into animal via posterior limb venous at a rate of 0.1～0.2ml/kg/min throughout the study.3. All 5 macaques were treated with somatostatin-14（Serono, Genevese, Switzerland） added to the normal saline solution at a dose of 5ug/kg/h iv prior to the onset of occluding superior mesenteric and through out 24 hours. Otherwise in control group, all animals received the same treatment except IIR operation.4. Tissues of ileum and pancrea were fixed in 4% buffered-paraformaldehyde solution and embedded in paraffin. Paraffin sections （5μn） were prepared and stained with haematoxylin and eosin. Injury was observed under macro- and microscopic examination while sections were scored with a semiquantitative scale designed to evaluate the degree of organ’ injury.5. SST concentration in peripheral intravenous blood was measured using a radioimmunoassay method.6. Ileum tissues and specimens of ileac epithelial cells, ileac lymphocytes, supernatant fluids ofileac lymphocytes incubation, peripheral blood, portal vein blood were obtained for detection the level of IFN-γusing ELISA.7. The concentration of tumor necrosis factor-alpha （TNF-alpha）, interleukin-1belta（IL-1β） and interleukin-6 （IL-6） in peripheral intravenous blood and Ileum tissues were measured using ELISA kit.8. The serum levels of amylase （AMY） and lipase were determined by chromometry.9. Immunohistochemistry were performed to identify the distribution of IFN-γ, CD4, CD8, CD57, CD20, CD5, PAX-5, plasma cell, TLR4, TLR2, NF-κB, integrinα4β7 and semi-quantitative immunohistochemical analyse was used to score integrated optical density（IOD） and ratio of area of object to total area of image（per area）.10. Blood culture ofpotal vein. 11. Human B lymphoblast cell line was cultured and divided into two groups. One is interferenced in medium supplemented with LPS （10μg/ml） and the other is control group. Stock cultures and the experimental cultures were maintained at 37℃in a moist atmosphere of 95% air and 5% CO₂.12. The concentration of IL-6 and IgM in culture cells supernatant fluid was determined by ELISA.13. The expression of TLR2 and TLR4 ofhmy2. cir was detected by immunofluorescence.14. MTT assay was used to measure the proliferation.Results1. IFN-γlevel in the ileac epithelial cells and potal vein blood after IIR were both remarkably increased （ 30.7±20.1 pg vs 9.4±2.4pg/mg total protein; 18.1±1.1 pg/ml vs 8.74±1.3 pg/ml）, p＜0.05; Whereas IFN-γlevel in the ileac lymphocytes and supematant fluid of that of IIR were reduced （1.7±0.02pg/mg vs 11.1±4.0pg/mg total protein; 2.2±0.3pg/mg vs 5.2±1.5pg/mg total protein）.2. After the injection of SST, IFN-γlevel decreased to 13.6±4.9pg/mg total protein, p=0.055 in the ileac epithelial cells, while in the potal vein blood to 8.9±2.5, p＜0.05. However, SST had no effect on IFN-γin the ileac lymphocytes and supernatant fluid.3. There is no change of IFN-γlevel in tissue homogenates and peripheral blood among three groups. Distribution of CD4⁺, CD57⁺ cells has no relationship with IFN-γ, while that of CD8⁺ cells demonstrated a conflicting tendency to IFN-γ.4. Numbers and areas of peyer’s patches were significantly increased postoperatively in IIR groups as compared to NC group.5. In peyer’s patches, the phenotype of B lymphocyte is PAX-5⁺CD20⁺CD5^- in NC groups deemed as B2 lymphocyte. In contrast, PAX-5⁺ B lymphocytes are increased （p＜0.05） and PAX-5⁺CD20^-D5^- B lymphocytes（pro-B cells） are observed in peyer’s patches of IIR groups. In NC group, plasm cells of mucosa locate mainly in lamina propria but decrease significantly in IIR group（p＜0.05）.6. In NC group, there is weak expression of TLR2, TLR4, NF-κB and IFN-γin peyer’s patches. IIR significantly increases the expression of the four molecules.7. Regarding toα4β7, there is sporadic expression in normal while IIR group has no expression in peyer’s patches.8. Compared to IL-1β（peripheral intravenous blood: 27.3±7.17ng/ml; ileac tissues: 82.8±20.5pg/g）、IL-6（peripheral intravenous blood: 13.9±10.50ng/ml; ileac tissues: 709.6±211.2pg/g）、TNF-α（peripheral intravenous blood: 3.04±1.01ng/ml; ileac tissues: 56.0±10.04pg/g）in normal group, these are increased in IIR group: IL-1β（peripheral intravenous blood: 79.2±14.4ng/ml; ileac tissues: 294.0±46.4pg/g）, IL-6（peripheral intravenous blood: 1261±297.5ng/ml; ileac tissues: 1527±160.8pg/g）, TNF-α（peripheral intravenous blood: 64.8±18.7ng/ml; ileac tissues: 213.2±29.2pg/g）significantly（p＜0.05）. Pretreatment of the macaques with somatostatin significantly inhibited the increased secretion of IL-1β（40.0±9.9 ng/ml）, IL-6（244.4±70.0 ng/ml）,TNF-α（19.2±10.1 ng/ml） compared with those of IIR group （p＜0.05）.9. In NC group, all blood cultures of potal vein were negative, whereas all animals’ speciments in IIR group were positively. Colon bacillus is the only bacterial translocation ingredient.10. Supernatant IgM levels（0.528±0.002 ng/ug total protein） were decreased 72 h after LPS treatment, with significant difference compared to control(（0.617±0.057ng/ug total protein） （p＜0.05）.11. Hmy2. cir cells showed a higher IOD of TLR4 （1201.3±913.4）and TLR2（174.8±80.2） 72 h after LPS stimulation compared with those of control group （TLR4: 5078.2±2483.2; TLR2: 1463.5±51.2） （p＜0.05）.12. LPS interference significantly decreases IL-6（0.528±0.002 pg/ug total protein） secretion from hmy2. cir cells compared to that of control group(0.406±0.218pg/ug total protein（p＜0.05）.13. Hmy2. cir cells displayed declined proliferation（0.389±0.061） after 24 h in response to LPS than that of control group（0.727±0.245）. 72 h after LPS treatment enhanced proliferation（0.562±0.219） was found in comparation to control group（0.173±0.079） （p＜0.05）.14. Activities of AMY （2 492.0±2 102.8 IU/L vs 385.4±136.1 IU/L） and lipase （1 278.0±1,446. 3 IU/L vs 25.8±13.0 IU/L） were increased （p＜0.05）. Pretreatment of the macaques with somatostatin significantly inhibited secretion of AMY（567.0±338.0 IU/L）and lipase（77.2±77.9 IU/L） in peripherial blood （p＜0.05）。15. After IIR injury, somatostatin leveI detected by RIA significantly decreased （62.17±13.56 pg/ml） in comparation to NC group（160.61±33.84 pg/ml） （p＜0.05） and got correct after SST treatment（156.32±30.25 pg/ml）（p＜0.05）.16. I1R group exhibited histopathologic injury of pancreas which was reduced from +++ to ++ after SST pretreatment.Conclusion1. After IIR, intestinal epithelial cells and pro-B lymphocytes produced a large number of IFN-γ, interacting with FcεR on mast cells in intestine, mucosa and tung where resulting in excessive innate immunity. In other words, IFN-γ, connected TLR- NF-κB pathway of innate immunity with mast cells. Thus it produced a sophisicated and highly active network of innate immunity.2. After IIR, pro-B lymphocytes in intestine participate in intestinal mucosal innate immunity. In addition, we hypothesize that serum or even marrow pro-B lymphocytes may be activated by LPS through TLR. Then is also involved in innate immunity and SIRS states of circulation by producing numerous IL-1β, IL-6, TNF-α. This is one of prominent causes of SIRS.3. After IIR, lack of rapid and vigorous response, PAX-5⁺CD20⁺ CD5^- memory B lymphoblast does not exhibit function of innate immunity.4. B lymphocytes are functional both in innate immunity and in acquired immunity but exert one immunomodulatory function at each stages of development. Pro-B cells may be activated by LPS through expression of TLRs. TLRs signal through NF-κB results in a rapid and vigorous innate immune response characterized by the production of pro-inflammatory cytokines; mature B cells has weak innate immune function but are central components of the adaptive immunity; the lymphoblast though expressing TLRs, does not participate in innate immunity because it is lack of activation function of LPS to TLRs.5. From IIR to MODS, pancreas is observed to damage in early stage. The inflammatory changes of pancreas after IIR are not induced by oxidative stress from oxygen deficiency, but are associated with NF-kB activation by inflammatory mediators in the circulation. Intravenous supplement of somatostatin shoud be benefitial in preventing the second hit to pancreas. It thus can avoid exacerbation of acute pancreatitis by suppressing systemic inflammatory reaction.更多还原