为了探索旋毛虫成虫期核酸酶家族Ts-DNase Ⅱ-7蛋白对Caco-2肠上皮细胞屏障模型的作用及机制,本研究采用Transwell细胞培养板构建Caco-2肠上皮细胞屏障,当跨膜电阻值(TEER)达到400 Ω·cm2即判定肠上皮屏障模型构建成功.将试验组分为对照组(control)、PBS组、Ts-DNaseⅡ-7组(5 μg/mL)、阳性对照LPS组(15 μg/mL),作用48 h后,检测跨上皮电阻值(TEER)和对FITC的通透性;Western-blot和荧光定量PCR(qPCR)检测各组细胞Claudin 1、Occludin和ZO-1的蛋白表达水平和mRNA表达水平;通过细胞免疫荧光观察各组紧密连接蛋白的分布情况.结果,与对照组相比,Ts-DNase Ⅱ-7组在第48小时TEER值降低,FITC的通透性增加;Claudin 1、Occludin和ZO-1的蛋白及mRNA的表达水平降低;免疫荧光试验可见细胞间的网状结构荧光不连续.结果表明,旋毛虫成虫期Ts-DNase Ⅱ-7蛋白可破坏肠上皮细胞的物理屏障,有助于旋毛虫实现顺利寄生.

目的复制肠道部分缺血再灌注(I/R)损伤动物模型.方法 90只大耳白兔随机分为失血性休克组、肠部分I/R组、假手术对照组、正常对照组.首先复制失血性休克动物模型,采用激光多普勒血流仪确定肠系膜上动脉(SMA)血流量的变化;其次,自行设计血流阻断器并测试其稳定性;最后,依据休克模型SMA血流量的变化,用自制的血流阻断器部分阻断SMA,复制肠道部分I/R损伤动物模型,同时观察多个脏器病理形态学和功能改变.结果兔失血性休克后,SMA血流量明显减少;SMA血流部分阻断50%,维持4小时,可造成兔肠道I/R损伤及多器官功能障碍.结论用自行设计的血流阻断器部分阻断SMA可成功复制出肠道部分I/R损伤动物模型.

<正>肠缺血再灌注(ischemia reperfusion,IR)损伤是感染、创伤、休克等常见疾病重要的病理生理环节,是临床众多疾病的基础。肠是体内代谢极活跃、具有独特免疫功能的器官,同时又是体内最大的致病菌库；20世纪90年代,肠道被认为是多器官功能障碍综合征(MODS)的启动器官。研究表明,肠IR可引起肠粘膜屏障受损,肠内细菌和内毒素移位,释放肠源性介质和预激循环白细胞,诱发全身炎症反应,甚至MODS。因而,肠IR损伤的研究受到高度重视,其发生

目的:探讨拟胆碱药卡巴胆碱对肠部分缺血再灌注损伤家兔血浆促炎细胞因子肿瘤坏死因子α、白细胞介素6和抗炎细胞因子白细胞介素10含量的影响. 方法:实验于2005-01/05在解放军第三○四医院完成.大耳白兔45只,随机分为卡巴胆碱治疗组20只、肠部分缺血再灌注组20只、假手术对照组5只.卡巴胆碱治疗组在部分阻断肠系膜上动脉2 h后肠内注射卡巴胆碱30 mg/kg.肠部分缺血再灌注组用自制血流阻断器阻断肠系膜上动脉血流50%,4 h后恢复灌流.假手术对照组不阻断肠系膜上动脉.各组分别于阻断前及阻断后2,4,8 h,1,2,3 d测定血浆促炎细胞因子肿瘤坏死因子α、白细胞介素6及抗炎细胞因子白细胞介素10的含量.结果:进入结果分析实验兔40只,卡巴胆碱治疗组18只、肠部分缺血再灌注组17只、假手术对照组5只.①家兔肠部分缺血再灌注损伤后,血浆肿瘤坏死因子α、白细胞介素6及白细胞介素10含量明显升高.②肠内注射卡巴胆碱后,血浆肿瘤坏死因子α含量在术后1 d显著降低,白细胞介素6含量在阻断4及8 h显著降低,与肠部分缺血再灌注组相比差异显著.③肠缺血再灌注组与卡巴胆碱治疗组血浆白细胞介素10含量相比无显著差异.结论:卡巴胆碱能明显抑制促炎细胞因子肿瘤坏死因子α和白细胞介素6的产生和释放,而对抗炎细胞因子白细胞介素10的影响甚微.表明拟胆碱药-卡巴胆碱具有明显的抗炎作用.

Aim: To investigate the effects of carbachol on the levels of the proinflammatory cytokines during partial ischemia-reperfusion(IR) of intestine injury in rabbits. Methods: White male big-ear rabbits(grade cleanness) were anaesthetized with soluble pentobarbitone via vein of ear margin, and then subjected to partial superior mesenteric artery occlusion(SMAO) (50%) for 4 hours, followed by reperfusion. The animals were randomly divided into three groups: carbachol group[carbachol (30 mg/kg) was injected into intestine 2 hours after partial SMAO], partial IR of intestine injury group, sham-operation control group. The levels of tumor necrosis factor-alfa (TNF-α) and interleukin-6(IL-6) in plasma were determined before partial SMAO and 2, 4, 6, 8 hours, 1 and 3 days after partial SMAO. The animals were sacrificed at 2, 6 hours, 1 and 3 days after partial SMAO, and then the pathological changes of the intestine tissue were fetched and observed. Results: The levels of TNF-α and IL-6 were significantly increased after partial IR injury. After injection with carbachol into intestine, the level of TNF-α was decreased significantly 1 day after operation, and the level of IL-6 was decreased significantly 4, 6 and 8 hours after SMAO in the carbachol group, compared with those in partial IR of intestine injury group. There were less inflammatory pathologic changes of the intestine issue after operation in the carbachol group than the partial IR of intestine injury group. Conclusion: Carbachol can inhibit the development and release of proinflammatory cytokines, and alleviate systemic inflammatory response during partial IR of intestine injury.

目的:研究兔肠道部分缺血再灌注(I/R)损伤过程中外周血中性粒细胞(PMN)凋亡的变化及机制.方法:大耳白兔55只,分为肠部分I/R组、假手术对照组、正常对照组,用自行设计的血流阻断器阻断肠系膜上动脉(SMA)血流50%,维持4h,制作肠道部分缺血再灌注操作动物模型:采用流式细胞术(AnnexinV/PI双染法)测定外周血PMN凋亡变化,采用放免法测定血浆内IL-6和TNF-α水平,采用免疫组织化学方法观察外周血PMNcaspase-3蛋白的表达,同时观察脏器功能和病理形态学改变.结果:肠部分I/R组,在肠道缺血期血浆IL-6T和TNF-α水平及外周血PMN凋亡比例均明显升高;在肠道再灌注期至术后1d外周血PMN凋亡比例骤然减少,而血浆IL-6和TNF-α在肠道再注期处于较高水平:外周血PMN caspase-3蛋白在肠道缺血期呈现高表达,而在再灌注期后表达明显降低.结论:肠道I/R全过程中PMN的凋亡与多种信号启动caspase-3蛋白的表达正相关.在肠道缺血期,IL-6及TNF-α可能是外周血PMN的凋亡过程的诱导分子或促进分子:而在肠道再灌注期,外周血PMN凋亡锐减可能是导致TNF-α、IL-6等细胞因子进一步增加的原因.

Objective To replicate an animal model of multiple organ dysfunciton syndrome(MODS)induced by partial ischemia of intestine,and to explore its mechanism.Methods Fifty white rabbits were randomly divided into 3 groups:sham operation group,ischemia and reperfusion(I/R) group,and hemorrhagic shock groups.The superior mesenteric artery(SMA)was partially occluded with a self-designed obturator to decrease SMA blood flow to 50% to 70%.The blood levels of GPT,Cr,CK-MB and TNF-α were measured from 0 h to 3days.The pathologic changes in different organs were also examed scales.Results When the SMA blood flow was reduced to(50.69±1.81)%,the levels of TNF-α,Cr,GPT,CK-MB increased dramatically.Severe pathological changes were observed in the heart,liver,and kidney.The mortality was 40%,and the incidence rate of MODS was 55%.Conclusion The etiological factor,the changes in organ funcitons,the pathological changes in different organs,the morbidity and mortality rates conform closely to early MODS after shock.The model can be used in further study of post-traumatic MODS.