【作者】 田轶伦；

【导师】 姜德谦；

【作者基本信息】 中南大学 ， 内科心血管， 2009， 博士

【摘要】 背景Fractalkine(FKN)是一具有粘附活性的趋化因子。膜结合型FKN独特的粘蛋白样杆状结构凸出细胞膜表面,使其易粘附流动的白细胞。并且,FKN介导的粘附反应不需要选择素(selectin)和整合素(integrin)等其他黏附分子的参与。FKN蛋白跨膜区N端有一段特殊的保守部位,即由Thr-Arg-Arg-Gln组成的二元断裂位点(dibasiccleavage site),在此断裂位点被蛋白水解酶水解脱落,形成可溶FKN,发挥趋化效应。FKN独特的结构和功能使其广泛参与炎症反应。慢性炎症反应是动脉粥样硬化(atherosclerosis,AS)的重要重要机制之一,白细胞越过血管向炎症部位移行是炎症反应中重要的生物学现象。单核细胞是AS中泡沫细胞的前体细胞,在向炎症部位迁移的过程中需要趋化因子和黏附分子的参与。近年来的研究资料表明,FKN参与了AS的发生发展。白细胞介素—18(Interleukin-18,IL-18)是重要的炎症因子,是动脉粥样硬化明确的危险因素。其在冠状动脉粥样硬化性心脏病的发生、发展及粥样斑块破裂等过程中发挥重要作用。以往的研究多认为,IL-18与AS的密切关系主要是通过干扰素-γ(interferon-γ,IFN-γ)实现的。而且,在IFN-γ缺失的情况下,IL-18的相关功能则无法体现。但近期的研究表明,在没有IFN-γ的参与下,IL-18也可以参与AS的炎症过程。IL-18与IL-18R结合导致多种与动脉粥样硬化相关的细胞因子如IL-6、IL-8、细胞黏附分子-1和多种基质金属蛋白酶(MMPs)分泌增加。然而,IL-18能否诱导和上调FKN的表达,并通过FKN的趋化和粘附功能参与炎症反应,及其机制尚不清楚。辛伐他汀(Simvastatine,Sim)是由土曲霉菌降解产物合成的三羟基三甲基戊二酰辅酶A还原酶抑制剂,广泛用于冠心病的预防和治疗。除降脂外,还具有改善内皮功能,改善心肌缺血,抗炎、抗血栓,稳定动脉粥样硬化斑块等作用。Sim能否通过影响FKN的表达而发挥抗炎、稳定斑块的作用尚不明确。目的(1)应用IL-18作为刺激因素,探讨IL-18能否诱导脐静脉内皮细胞(HUVEC)上调表达FKN,明确两者的作用关系及信号通路;(2)应用Transwell进行体外趋化试验,观察FKN对巨噬细胞源性单核细胞株(THP-1)的趋化作用;(3)应用Flow chamber模拟人体生理血流环境,观察FKN介导的HUVECs和THP-1细胞的粘附;(4)探讨辛伐他汀对FKN表达的影响,以及对FKN趋化和粘附作用的干预效应。方法(1)HUVECs与100 ug/L的IL-18孵育0、1、6、12、24小时,半定量RT-PCR方法检测FKN的mRNA表达;(2)HUVECs分别与浓度为0ug/L、25 ug/L、50 ug/L、100 ug/L的IL-18共孵育24小时,半定量RT-PCR方法检测FKN的mRNA表达;(3)预先用10umol/L、30umol/L的PDTC预处理HUVECs,再用100ug/L的IL-18刺激24小时,半定量RT-PCR方法检测FKN的mRNA表达;(4)预先用1umol/ml、10umol/ml、100umol/ml的辛伐他汀预处理HUVECs,再用100ng/L的IL-18刺激24小时,半定量RT-PCR方法检测细胞FKN的mRNA表达;(5)HUVECs与0ug/L、25 ug/L、50 ug/L、100 ug/L的IL-18孵育24小时;10umol/L、30umol/L的PDTC预处理HUVECs,再用100ug/L的IL-18刺激24小时;预先用10umol/ml、100umol/ml的辛伐他汀预处理HUVECs,再用100ng/L的IL-18刺激24小时;100 ug/L的IL-18刺激HUVECs 24小时后,用1umg/L、5ug/L的FKN中和抗体作用3小时。应用Transwell装置观察FKN对THP-1细胞的趋化效应;(6)HUVECs与0ug/L、25 ug/L、50 ug/L、100 ug/L的IL-18孵育24小时;10umol/L、30umol/L的PDTC预处理HUVECs,再用100ug/L的IL-18刺激24小时;预先用10umol/ml、100umol/ml的辛伐他汀预处理HUVECs,再用100ng/L的IL-18刺激24小时;100 ug/L的IL-18刺激HUVECs 24小时后,用1umg/L、5ug/L的FKN中和抗体作用3小时。应用Flow chamber装置观察FKN在模拟生理血流环境下介导的HUVEC与THP-1细胞的粘附。结果(1)HUVECs与100ug/L的IL-18孵育1小时后,FKN表达无明显增加,与空白对照组相比p＞0.05;HUVECs与100ug/L的IL-18孵育6、12、24小时后,FKN的基因表达明显增加,与空白对照组比较p＜0.01;(2)HUVECs与5ug/L的IL-18孵育24小时后,FKN表达量无明显增加,与空白对照组相比p＞0.05;HUVECs与25 ug/L、50 ug/L、100 ug/L的IL-18孵育24小时后,FKN的基因表达明显增加,与空白对照组比较p＜0.01;(3)用10umol/L、30umol/L的PDTC预处理HUVEC后,再加入100ug/L的IL-18孵育24小时,FKN表达量明显减少,与未经PDTC处理的单纯100ug/L IL-18刺激组比较p＜0.01;(4)用1 umol/L的辛伐他汀预处理HUVECs后,再加入100ug/L的IL-18孵育24小时,FKN表达量无明显变化,与单纯100ug/L IL-18刺激组比较p＞0.05;用10umol/L、100umol/L辛伐他汀预处理HUVEC后,再加入100ug/L的IL-18孵育24小时,FKN表达量明显减少,与未经辛伐他汀处理的单纯100ug/L IL-18刺激组比较p＜0.01。(5)成功使用Tranwell装置进行了体外趋化试验;FKN能够趋化静息的THP-1细胞;(6)不同浓度IL-18上调FKN表达后,对THP-1细胞的趋化效应明显增强,与对照组比较p＜0.01;PDTC、辛伐他汀预处理HUVEC后,该趋化效应明显减弱,与单纯IL-18刺激组相比p＜0.01;FKN中和抗体能够抑制FKN介导的趋化作用,与单纯IL-18刺激组相比p＜0.01。(7)成功应用Flow chamber在体外模拟了人体生理状态的血液循环条件进行粘附实验;使用EDTA阻断其他黏附分子的参与后,FKN以非钙离子依赖的方式介导了HUVECs与单核细胞THP-1的粘附;(8)不同浓度IL-18上调FKN表达后,THP-1细胞与HUVEC的粘附明显增加,与对照组比较p＜0.01;PDTC预处理HUVEC后,该粘附效应明显减弱,与单纯IL-18刺激组相比p＜0.01。FKN中和抗体能够抑制FKN介导的粘附作用,与单纯IL-18刺激组相比p＜0.01。10umol/ml的辛伐他汀预处理HUVEC后,未能明显抑制FKN介导的粘附反应,与单纯IL-18刺激组相比p＞0.05。100umol/ml的辛伐他汀能够明显抑制该粘附效应,与单纯IL-18刺激组相比p＜0.01。结论(1)IL-18能够呈浓度、时间依赖性上调FKN的基因表达。这种诱导作用与NF-KB信号转导途径有关;(2)FKN能够趋化静息THP-1细胞,IL-18能够增加THP-1细胞的迁移;(3)在模拟生理血液循环的条件下,FKN能够介导HUVECs与静息THP-1细胞的粘附,IL-18能够增强该粘附反应;(4)辛伐他汀能够抑制IL-18诱导HUVECs上调表达FKNmRNA,抑制FKN介导的趋化及粘附作用。更多还原

【Abstract】 BackgroundFractalkine（FKN） is a membrane-tethered chemokine that functions as chemokines and adhesion molecules to capture and induce firm adhesion of a subset of leukocytes in a selectin- and integrin -independent manner.Soluble FKN can be released,presumably by proteolysis at a membrane-proximal dibasic cleavage site（Thr-Arg- Arg-Gln）,and exhibits an efficient chemotactic activity for monocytes.The novel structure and functions of FKN suggest that it may play a key role in the multistep process of leukocyte trafficking,while raising several issues. Chronic inflammation is one of the mechanism of Atherosclerosis （AS）.The migration of leukocyte through intima is an essential biological phenomena of inflammation.Monocytes are important cells for forming the foam cells.The primary step of this is to migrate through intima too.The molecular control of these essential trafficking events requires two broad classes of molecules:various cell adhesion molecules,as well as leukocyte chemotactic factors,the chemokine superfamily.Now we have increasingly more evidence that FKN participate in the formation and development of atherosclerosis（AS）.In recent years,increasing evidences has emerged from experimental and epidemiological data that interleukin- 18（IL- 18）,a pro-inflammatory cytokinee involved in both innate and acquired immune responses,plays a key role in the inflammatory response that contributes to AS.Baseline circulating IL-18 levels were strongly predictive of future cardiovascular disease.Most past studies suggest that IL-18 function mainly through interferon-γ（IFN -γ）,and could not intervene AS without IFN-γ.Recent studies make it clear that IL-18 could also mediate several reactions in a IFN -γindependent manner.Banding of IL-18 and IL-18 receptor enhances the secretion of IL-6、IL-8、ICAM-1 and MMPs.However,it remains unclear whether IL-18 up-regulate the expression of FKN,and participate AS through the chemotactic and adhesive function of FKN.Simvastatin is the 3-hydroxy-3-methylglutanyl coenzyme A（HMG-CoA） reductase inhibitor lipid-lowering agents synthesized from degradation products of aspergillus terreus.It has been used as one of the important medicine for coronary heart diseases.Besides lipid-lowering function,Simvastatin could protect endothelial cells,improve myocardial ischemia,anti-inflammatory anti-thrombosis and stabilize atherosclerotic plaques.However,it is still unclear whether Simvastatin exert its role through effect the expression and chemotactic,adhesive function of FKN.Objective（1）To observe the expression of FKN induced by IL-18 in HUVECs, and to investigate its signal pathway.（2）Using Transwell to observe the chemotaxis of FKN and the effect of IL- 18 on monocyts migration; （3）Using in vitro Flow chamber mimic the vivo fluid dynamic environment to observe the adhesion between HUVECs and THP-lcells mediated by FKN and the effect of IL-18;（4）To investigate the effect of Simvastatine on expression of Fractalk -ine upregulated by IL-18,and the interventional effect on chemotaxis, adhesive function of FKN.Methods（1） HUVECs were cultured and treated with IL-18 of concentrations 100μg/L for 0,1,6,12,24 hours.FKN mRNA expressions were determined by semi-quantitative reverse-transcription polymerase chain reaction（RT-PCR）.（2） HUVECs were cultured and treated with IL-18 of 4 different concentrations（5μg/L,25μg/L,50μg/L,and 100μg/L） for 24 hours.We determined mRNA expressions of FKN by semi-quantitative reverse-transcription polymerase chain reaction（RT-PCR）.（3） Pretreated HUVECs with PDTC（A specific inhibitor of NF-KB） of 2 different concentrations（10umol/L,30umol/L）,then cells were treated with IL-18（100ug/L） for 24 hours,mRNA expressions of FKN were determined by semi-quantitative RT-PCR.（4）Pretreated HUVECs with Simvastatine of 3 different concentrations （1 umol/L,10umol/L,100umol/L）,then cells were treated with IL- 18 （100ug/L） for 24 hours.FKN mRNA expressions were determined by semi-quantitative RT-PCR.（5） HUVECs were treated as above 1,3,4 method.The other HUVECs were cultured and treated with IL- 18 of 100 ug/L for 24 hours, then cells were treated with neutralizing antibody of FKN（1 ug/L,5ug/L） for 2 hours.The migration of THP-1 cells mediated by FKN was odserved by Transwell.（6） HUVECs were treated as above 1,3,4 method.The other HUVECs were cultured and treated with IL- 18 of 100 ug/L for 24 hours, then cells were treated with neutralizing antibody of FKN（1ug/L,5ug/L） for 2 hours.The adhesion of HUVECs and THP-1 cells mediated FKN was odserved by In vitro Flow chamber assay.Results（1） HUVECs cultured and incubated with 100ug/L IL- 18 for 1 hour resulted in no difference of FKNmRNA expression as compared with control group（p＞0.05）;HUVECs were cultured and incubated with IL-18 of concentrations 100μg/L for 6,12,24 hours resulted in upregulated expression of FKNmRNA,as compared with control group （p＜0.01）;（2） HUVECs cultured and incubated with 5ug/L IL-18 for 24 hours resulted in no difference of FKNmRNA expression,as compared with control group（p＞0.05）;HUVECs were cultured and incubated with IL-18 of concentrations 25,50,100μg/L for 24 hours resulted in upregulated expression of FKNmRNA,as compared with control group（p＜0.01）;（3） Pretreated HUVECs with PDTC of 10umol/L,30umol/L and then incubated with IL-18（100ug/L） for 24h resulted in downregulated expression of FKN mRNA,as compared with the pure IL-18（100ug/L） group（p＜0.01）;（4） Pretreated HUVECs with Simvastatin of 1umol/L and then incubated with IL-18（100ug/L） for 24h resulted in no difference of FKNmRNA expression,as compared with the pure IL-18（100ug/L） group （p＞0.05）;Pretreated HUVECs with Simvastatin of 10umol/L and 100umol/L and then incubated with IL-18（100ug/L） for 24h resulted in down-regulated expression of FKN mRNA,as compared with the pure IL-18（100ug/L） group（p＜0.01）.（5）The chemotactic assay was successful by using Transwell,FKN could chemotaxis the resting THP-1 cells;（6） After up-regulated FKN mRNA expression induced by IL-18 with different concentrations,the chemotactic effect of FKN to THP-1 cells were enhanced significantly,consistent to the expression of FKN mRNA.The chemotactic effect were inhibited if HUVECs pretreated with Simvastatin or PDTC,or treated with neutralizing antibody,as compared with the pure IL-18（100ug/L） group（p＜0.01）（7）Using in vitro flow chamber mimic the vivo fluid environment secessfully,FKN could mediate the adhesion in a Ca²⁺ independent maner;（8） After up-regulated FKN mRNA expression induced by IL- 18,the adhesion mediated by FKN was enhanced significantly,as compared with control group（p＜0.01）.The adhesion were inhibited after HUVECs pretreated with PDTC,or treated with neutralizing antibody,as compared with pure IL-18（100ug/L） group（p＜0.01）.Simvastatin with 10umol/ml concentration did not inhibit the adhesion,as compared with the pure IL-18（100ug/L） group（p＞0.05）.Simvastatin with concentration of 100umol/L inhibated the adhesion,as compared with the pure IL- 18（100ug/L） group（p＜0.01）.Conclusions（1） IL-18 could up-regulate the expression of FKN mRNA in a dose-and time-dependent manner,and this effect might be related to NF-KB.（2） IL-18 could enhance the chemotactic and adhesive effect of HUVECs to THP-1 cells significantly by up- regulation of FKN.（3） Simvastatin could down-regulated the expression of FKN mRNA induced by IL-18,and inhabit the chemotactic and adhesion.更多还原