【作者】 徐志宏；

【导师】 施小燕；

【作者基本信息】 浙江大学 ， 急诊医学， 2008， 硕士

【摘要】 背景及目的儿茶素单体表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)是绿茶多酚类化合物的一种,具有抗脂质过氧化、清除自由基、抗突变等生物学活性。以往大量实验证实了EGCG等一系列儿茶素单体在肿瘤化学预防方面的功效,认为其参与调节肿瘤发生的细胞信号通路中氧化还原敏感的转录因子的活性。其中核因子κB(NF-κB)和激活蛋白1(AP-1)为各种炎症和肿瘤的发生侵袭过程共同涉及的关键转录因子。故而儿茶素在肿瘤化学预防上的大量研究同时揭示了该类化合物在抗炎治疗方面的巨大潜力。儿茶素类在动脉硬化、类风湿性关节炎等慢性炎症性疾病中已经被证明了具有积极的功效,EGCG更被证明为对炎症细胞因子合成影响最显著的儿茶素单体。但是相对于慢性炎症,EGCG对急性炎症过程的干预研究还不多。全身性炎症反应综合征(Systemic Inflammatory Response Syndrome,SIRS)是多器官功能不全综合征(Multiple Organ Dysfunction Syndrome,MODS)的重要病理生理发展通路。外周血多形核中性白细胞(polymorphonuclear neutrophilsw,PMNs)是引起组织损伤,甚至MODS的关键细胞。PMNs激活、聚集于靶组织和释放大量破坏性酶类是导致组织损伤和MODS发生的主要途径,在此过程中免疫趋化因子和细胞间粘附分子发挥了重大作用。局部组织合成的趋化因子,以及PMNs/内皮细胞间的稳定粘附过程,对于PMNs的贴壁、游出、定向移动和大量靶器官“扣押”具有不可替代的重要作用。本课题结合革兰氏阴性菌内毒素脂多糖(Lipopolysaccharides,LPS)诱导的小鼠急性肺损伤模型和TNF-α诱导的内皮细胞炎症模型进行一系列体内体外研究,旨在探讨EGCG作为一种高效价的天然抗氧化剂,在全身性炎症反应早期对组织巨噬细胞合成趋化因子的影响,以及对PMNs/血管内皮细胞间稳定粘附的影响;并对以髓质过氧化物酶(myeloperoxidase,MPO)活力代表的肺组织PMNs浸润程度作出评估,从而多方面地论证EGCG对SIRS早期PMNs远隔脏器浸润的影响。实验方法1.实验动物和细胞的准备6周龄ICR小鼠,清洁级,EGCG分别以10mg/kg、20mg/kg、40mg/kg的剂量行腹壁多点皮下注射预处理,以尾静脉注射LPS 10mg/kg方法造成内毒素血症,复制全身性炎症反应动物模型。根据研究的需要分别在给予LPS6小时和18小时后处死小鼠,留取肺组织标本。传代人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)用含10%胎牛血清的DMEM高糖培养基,置于37℃、5%CO2的细胞培养箱中培养。贴壁生长至80%板底面积的HUVCE分别换用含EGCG终浓度为10μmol/l、20μmol/l、50μmol/l的新鲜培养基行1小时预处理,然后加入TNF-α使终浓度为10ng/ml,复制出内皮细胞炎症模型。根据研究的需要分别在给予TNF-α4小时和8小时后消化收集细胞用于指标检测。2.RT-PCR法分析小鼠巨噬细胞炎症蛋白2(murine macrophage inflammatoryprotein 2,MIP-2)和ICAM-1的基因表达水平在LPS攻击6小时后处死各组小鼠,无菌留取肺组织;消化收集经EGCG预处理和TNF-α诱导4小时后的HUVEC。TRIZOL法提取总RNA,逆转录后PCR扩增,分别以小鼠β-Actin和人GAPDH为内参照行半定量分析。3.免疫组化染色及形态学观察在LPS攻击18小时后处死各组小鼠,留取肺组织固定,石蜡包埋,切片,以抗小鼠MIP-2多克隆抗体为一抗,辣根过氧化酶标记相应二抗结合,DAB显色,苏木素复染镜检,拍照。4.Western Blot法分析MIP-2和ICAM-1的蛋白表达水平在LPS攻击18小时后处死各组小鼠,留取肺组织匀浆裂解;消化收集经EGCG预处理和TNF-α诱导8小时后的HUVEC。提取总蛋白,行SDS-PAGE电泳,转膜,孵育以相应一抗、二抗,ECL发光液,曝光成像,以β-Actin为内参照对目的条带的积分吸光度进行分析。5.肺组织MPO活力测定在LPS攻击18小时后处死各组小鼠,留取肺组织,准确称量并匀浆,利用MPO令供氢体邻联回香胺供氢后生成黄色化合物在460nm处有最大吸收峰的化学特性,用分光光度法检测。按MPO检测试剂盒说明操作和换算。6.噻唑兰(MTT)试验鉴定HUVEC细胞活力贴壁长满75%的96孔板底面积的HUVEC,经不同浓度EGCG预处理和TNF-α诱导8小时后更换含MTT终浓度5mg/ml的新鲜培养基继续孵育4小时,吸弃上清加入二甲基亚砜(DMSO),继续孵育半小时,酶标仪490nm波长处测OD值。7.HUVEC跟PMNs的粘附实验取健康成年人外周血10ml,用中性粒细胞分离液(聚蔗糖—泛影葡胺分层液)以密度梯度离心法得到较纯的PMNs。台盼蓝拒染法检测活力,血清培养基重悬PMNs。贴壁生长在24孔板底的HUVEC经EGCG预处理和TNF-α诱导8小时后加入等量PMNs,再共孵育1小时,结束后轻洗去未粘附的PMNs,收集洗脱液,对孵育前和洗脱液中的PMNs进行细胞计数,计算出粘附率。8.数据统计数字结果以均数±标准差表示,多组间结果比较采用单因素方差分析,P＜0.05认为有统计学显著性差异。数据的统计处理使用SPSS10.0版完成。结果1.EGCG使内毒素诱导的急性肺损伤小鼠肺部MIP-2的mRNA表达和蛋白合成呈剂量依赖性下降,差异具有统计学意义(P＜0.05)。2.免疫组化和组织病理发现EGCG使内毒素诱导的急性肺损伤小鼠肺部MIP-2蛋白表达减少,并且使肺间质肿胀和白细胞浸润情况减轻。3.EGCG使内毒素诱导的急性肺损伤小鼠肺部MPO活力呈剂量依赖性下降,差异具有统计学意义(P＜0.05)。4.50μmol/l的EGCG浓度导致了TNF-α诱导的HUVEC和正常生长的HUVEC的凋亡增加(P＜0.05),而10μmol/l、20μmol/l两个浓度的EGCG不引起HUVEC的活力改变(P＞0.05)。5.EGCG使TNF-α诱导的HUVEC的ICAM-1的mRNA表达和蛋白合成呈剂量依赖性下降,差异具有统计学意义(P＜0.05)。6.EGCG使TNF-α诱导的HUVEC跟PMNs间的粘附率呈剂量依赖性下降,差异具有统计学意义(P＜0.05)。结论本研究在动物实验中发现EGCG抑制内毒素诱导全身炎症反应的小鼠肺部趋化因子的合成以及减少肺组织PMNs的浸润程度,在细胞实验中发现EGCG抑制TNF-α诱导的HUVEC膜表面重要粘附分子的合成以及抑制HUVEC和PMNs间的粘附,并发现这些效应呈现一定的浓度和剂量依赖性。故而本研究提示抗氧化剂EGCG对SIRS早期的免疫粘附和趋化过程具有抑制作用,从而可能对后期的靶器官功能损伤具有积极的预防意义。更多还原

【Abstract】 Background and ObjectiveEpigallocatechin-3-gallate（EGCG）,which is known as catechin monomer,is a kind of tea polyphenols compounds and has the biologic activities of anti-lipid peroxidation, cleaning of free radical and antimutation.A great quantity of former experiments have validated the effectiveness of a series of catechin monomers,such as EGCC,on the regards of chemical prophylaxis of tumors,as they participate to regulate the activity of oxidoreduction-sensitive transcription factor in tumorigenic cell signal pathway.And nuclear factorκB（NF-κB）and activator protein 1（AP-1）are the key transcription factors, which are commonly related in the process of development and invasion of various inflammations and tumors.Thus,generous researches about catechin on chemical prophylaxis of tumors have simultaneously revealed its great potential in anti-inflammatory treatment.Catechin family has been proved to have the positive efficacy in chronic inflammation diseases such as arteriosclerosis and arteriosclerosis. And EGCG has also been proved to be the catechin monomer which has the most conspicuous influence upon composition of inflammatory cytokines,but there are not much studies on intervention in acute inflammatory process,opposite to chronic inflammation.Systemic Inflammatory Response Syndrome（SIRS）is the important pathologic and physiologic developing pathway of Multiple Organ Dysfunction Syndrome（MODS）. Polymorphonuclear neutrophilsw（PMNs）is the key cell which causes tissue damages, even MODS.PMNs’ activation,aggregation at target tissues and liberation of large amounts of destructive enzymes is the main path which induces tissue damages and MODS.In this process,immune chemotatic factors and intercellular adhesion molecules have played important roles.Chemotatic factors composited by regional tissues and stable adhesion between PMNs and endothelial cells（ECs）have their great irreplaceable contributions to PMNs’ adherence,emigration,directional migration and considerable sequestration into target organs.Combining the lipopolysaccharides（LPS）-induced mouse acute lung injury model and TNF-α-induced endothelial cell inflammation model,this study carries out a series of researches in vivo and vitro,in order to investigate the effects of EGCG,as a natural anti-oxidant with high potency,upon synthesis of chemotatic factors,and upon stable adhesion of PMNs/ECs,at the early phase of systemic inflammation.The thesis also evaluates the infiltration degree of PMNs in lung,which is represented by the activity of myeloperoxidase（MPO）,thereby explores the EGCG’s effect upon PMNs infiltration to remote organs at the early phase of SIRS in several aspects.Methods1.Animals preparation and cell culture6-week old ICR mice of clean grade,were pretreated by EGCG hypodermic injection at abdominal walls with doses of 10mg/kg,20mg/kg or 40mg/kg.Endotoxemia was caused by means of vena caudalis LPS injection with dose of 10mg/kg.The animal model of systemic inflammatory response was duplicated.The mice were executed respectively 6h and 18 h after LPS challenge according to the study need and lung samples were preserved. Human umbilical vein endothelial cells（HUVEC）were cultured in high glucose DMEM supplemented with 10%foetal bovine serum（FBS）and placed in an incubator under condition of 37℃and 5%CO₂.The medium were removed and replaced respectively by fresh one with EGCG final concentration of 10μmol/1,20μmol/1 and 50μmol/1,when HUVEC were adheringly cultured enough to cover 80%bottom area.After 1 h pretreatment,TNF-αwere put in with final concentration of 10 ng/ml and the model of endothelial cell inflammation was duplicated.According to the study need,after 4 h and 8 h TNF-αadministration,cells were respectively digested and collected for detections.2.To analyze the gene expression levels of murine macrophage inflammatory protein 2（MIP-2）and ICAM-1 through RT-PCRAfter 5 h LPS attack,different groups of mice were executed and lungs were reserved. HUVEC were collected after EGCG pretreatment and 4 h TNF-αinduction.Total RNA was extracted by TRIZOL.After reverse transcription,PCR and semiquantitative analysis were performed,taking miceβ-Actin and human GAPDH as reference.3.Immune histochemistry dyeing and morphology observationAfter 18 h LPS attack,different groups of mice were executed and lungs were reserved and fixed.Then the tissus were paraffin imbedded,sliced and incubated in anti-mouse MIP-2 polyclonal antibody and HRP labeled IgG successively,with DAB coloration,hematoxylin afterstain test under microscope and taking photos.4.To analyze the protein expression levels of MIP-2 and ICAM-1 through Western BlotAfter 18 h LPS attack,different groups of mice were executed and lungs were reserved,homogenated and split.HUVEC were collected after EGCG pretreatment and 8 h TNF-αinduction.Total protein was extracted,performed SDS-PAGE,and transferred onto PVDF membrane,which was incubated with the fast antibody,the second antibody, and ECL mixture successively.Then the membrane was processed by exposal and develop.The integral absorbance of the bands were measured withβ-Actin as reference. 5.The metry on MPO vigor of lung tissusAfter 18 h LPS attack,different groups of mice were executed and lung tissus were reserved and weighed accurately.The yellow compound which is the remains of O-dianisidine after the donation caused by MPO has the maximum absorption peak at the point of 460nm.The products were detected by spectrophotometric method,according to the manufacturer’s instructions of the MPO detecting kit.6.The viability of HUVEC determined by MTT assayWhen adheringly cultured enough to cover 75%bottom area of 96-well plant, HUVEC were processed by different concentrations of EGCG pretreatment and 8 h TNF-αinduction.Then the medium were removed and replaced by fresh one with MTT. After 4 h incubation,the supernatant was abandoned and the DMSO was added in.After 30 m incubation,the OD value was determined at the wave length of 490nm.7.The adhesion tests of HUVEC and PMNs10ml peripheral blood of a healthy adult was drawn and treated with demixing effusion of Ficoll-Hypaque by density gradient centrifugation,to produce purer PMNs. The viability of the PMNs were determined.The partes aequales PMNs were added in the HUVEC which were adheringly cultured at the bottom of 24-well plant,after EGCG pretreatment and 8 h TNF-αinduction.After 1 h incubation,PMNs which were not adherent were rinsed and the eluant was collected.PMNs before the incubation and in the eluant were counted respectively and the adhesion ratios were calculated.8.Statistical evaluationThe results were expressed as mean±S.D.;Statistical comparisons were made among groups using a one-way analysis of variance.P-value less than 0.05 was considered to be statistically significant difference.SPSS10.0 was used for statistical processing of data.Results1.EGCG made the mRNA expressions and protein synthesis of MIP-2 descend dose-dependently in mice lungs with endotoxin-induced acute lung injury.The differences had statistical significance（P＜0.05）.2.Immune histochemistry and histopathology demonstrated that EGCG made the protein expressions of MIP-2 decrease in mice lungs with endotoxin-induced acute lung injury, and lessened the situation of engorgement in lung interstitial tissues and Leukocytic infiltrate.3.EGCG made the MPO vigor descend in mice lungs with endotoxin-induced acute lung injury dose-dependently.The differences had statistical significance（P＜0.05）.4.EGCG with the concentration of 50μmol/1 led to the increasing of Apoptosis of TNF-α-induced HUVEC and normal growing HUVEC（P＜0.05）,while EGCG with the concentration of 10μmol/1 and 20μmol/1 did not result in the changing of HUVEC’s viability（P＞0.05）.5.EGCG made the mRNA expressions and protein synthesis of ICAM-1 descend in TNF-α-induced HUVEC dose-dependently.The differences had statistical significance （P＜0.05）.6.EGCG made the ratios of adhesion between TNF-α-induced HUVEC and PMNs descend dose-dependently.The differences had statistical significance（P＜0.05）.ConclusionThe study in vivo finds that EGCG dose-dependently inhibits the synthesis of chemotatic factors in mice’s lungs with endotoxin-induced systemic inflammatory reaction and reduces the infiltration degree of PMNs in lung.And the experiments in vitro finds that EGCG dose-dependently inhabits the TNF-α-induced synthesis of the important adhesion molecules on the surface of HUVEC membrane as well as the adhesion between HUVEC and PMNs.Consequently,the experiment has certified that anti-oxidant EGCG possesses the inhibitive effect on the immune adherence and chemotaxis at the early phase of SIRS and accordingly may have the positive precaution significance on the function damage of target organs at the late phase.更多还原