摘要
目的
他汀类(Statins)药物属于3-羟-3-甲基戊二酰辅酶A还原酶(HMG-CoA)抑制剂,可以降低血清胆固醇的含量,是治疗高脂血症和动脉粥样硬化的药物中最有效的一类。越来越多的研究表明,他汀类药物降低心血管疾病的发病率和死亡率不仅仅依靠其降脂作用,除此以外还有多种药理作用,如改善血管内皮、促进纤溶、抗血栓形成等等。近年来,有关他汀类药物抗炎作用的研究日渐增多,其抗炎作用的发现表明,在临床中应用他汀类药物治疗动脉粥样硬化除依靠降脂作用外还有其他的益处,但是目前具体的抗炎机制尚不明确。
白细胞介素6、8(interleukine6,8 IL-6,IL-8)、肿瘤坏死因子α(tumornecrosis factorαTNFα)、尿激酶型纤溶酶原激活物及其受体(urokinaseplasminogen activator/urokinase plasminogen activator receptor,uPA/uPAR)均可由内皮细胞分泌和表达,在炎症反应中,血管内皮细胞受刺激或损伤后可以导致这几种炎症介质的表达和释放增多,从而激活和趋化白细胞、介导白细胞和内皮细胞的黏附、促进白细胞的迁移等,因此,IL-6、TL-8、TNFα、uPA/uPAR在炎症的发生发展过程中扮演重要的角色。
本次实验利用炎症刺激因子—脂多糖(Lipopolysaccharides,LPS)诱导人脐静脉内皮细胞(Human umbilical vein endothelial cell,HUVEC)炎症介质IL-6、IL-8、TNFα、uPA/uPAR的分泌和表达,并观察辛伐他汀对几种炎症介质表达的影响,从而为进一步研究他汀类药物的抗炎作用机制提供理论依据。
方法
1.细胞培养:取新鲜脐带,分离人脐静脉内皮细胞,培养,鉴定,传代,第2-3代的细胞用来实验。
2.细胞分组:将细胞随机分成4组:①对照组;②LPS组(100ng/ml);③辛伐他汀组(5μg/ml);④LPS+辛伐他汀组。细胞分别处理1h、12h、24h后,取细胞上清液做ELISA检测,同时将细胞冻存留做荧光实时定量PCR检测。
3.取细胞上清液,分别做IL-6、IL-8、TNF-α的ELISA检测,纪录结果。
4.收集细胞,分别抽取mRNA,反转录cDNA,进行荧光实时定量PCR检测IL-6、IL-8、TNF-α以及uPA/uPAR的mRNA表达,纪录结果。
5.上述实验重复3次,并用统计学分析软件进行统计分析。
结果
1.人脐静脉内皮细胞鉴定结果:形态学鉴定,相差显微镜下,内皮细胞分布均匀,单层生长,形态为多角形,鹅卵石状镶嵌排列;内皮细胞Ⅷ因子相关抗原免疫荧光检测鉴定,荧光显微镜下,内皮细胞胞浆中可见大量黄绿色荧光。鉴定结果为人脐静脉内皮细胞。
2.ELISA结果:对照组细胞,在不施加任何刺激的情况下可以少量分泌IL-6、IL-8、TNF-α;LPS刺激组(100ng/ml)细胞,IL-6、IL-8、TNF-α的分泌量比对照组明显增加,且有显著性差异(p＜0.01);辛伐他汀与LPS共孵育组IL-6、IL-8、TNF-α分泌量明显比LPS刺激组减少,且有显著性差异(p＜0.01)。
3.实时定量PCR结果:LPS刺激组(100ng/ml)细胞,IL-6、IL-8、TNF-α以及uPA/uPAR的mRNA表达比对照组明显增强,且有显著性差异(p＜0.05);辛伐他汀与LPS共孵育组IL-6、IL-8、TNF-α以及uPA/uPAR的mRNA表达明显比LPS刺激组减少,且有显著性差异(p＜0.05)。
结论
1.LPS可以诱导人脐静脉内皮细胞IL-6、IL-8、TNF-α、uPA/uPAR mRNA表达增多。
2.辛伐他汀可以有效抑制由LPS诱导的人脐静脉内皮细胞IL-6、IL-8、TNF-α、uPA/uPAR mRNA表达。
Objective:
Statins are potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase.These drugs are capable of lowering the serum levels of cholesterol and are successfully used to treat hypercholesterolemia and atherosclerosis.Moreover,the ability of statins to reduce the mortality and morbidity of cardiovascular diseases has been ascribed not only to their cholesterol-lowering activities but also to a number of additional effects,including improved endothelial cell function,enhanced fibrinolysis, and antithrombotic activity.In addition,a number of important anti-inflammatory effects of statins have been reported.The anti-inflammatory effects of statins implicated more clinical benefit that can be obtained in the treatment of atherosclerosis.Little is known about the mechanisms by which statins counteract inflammation.
Interleukine 6(IL-6)、interleukine 8(IL-8)、tumor necrosis factor (TNFα)、urokinase plasminogen activator/urokinase plasminogen activator receptor(uPA/uPAR)are essential inflammatory mediators which play important roles in the inflammation process.They can be expressed from endothelial cells which induced by injurious factors to active the leukocyte and infhence the process of chemotactic、adhesion and transmigration.
We use the inflammatory stimulating factor-Lipopolysaccharides (LPS)on the human umbilical vein endothelial cell(HUVEC)to induce the mRNA exprssion of IL-6、IL-8、TNFαand uPA/uPAR.Then we observe the influence of simvastatin on the mRAN expression of HUVEC.The results can be used to improve the theory of inflammatory mechanism of statins.
Method:
1.Cell culture:Collect the fresh umbilical core,cultivate the umbilical vein endothelial cells,identified,passage,the cells of 2-3 era could be use.
2.Divide into four groups random:①cntrol group;②LPS group(100ng/ml);③simvastatin group(5μg/ml);④LPS and simvastatin group(10μg/ml).The four groups of cells were co-incubated for 1-24 hours(1h,12h,24h).
3.The levels of IL-6,IL-8,TNFαin the collected culture supernatants were measured by the ELISA kit.
4.The mRAN expression of IL-6,IL-8,TNFαand uPA/uPAR of the HUVEC were detected by the real-time quantitative PCR(RT-PCR).
5.The experiment above were repeated three times,then use software to do the statistical anlysis.
Result:
1.The identification of HUVEC:The morphology identification,in the microscope,the endothelial cells distribute uniformity,grow in monolayer,polygon,arrays like cobblestone-appearance.FactorⅧantigon immunofluorescence detection,in the fluorescent microscope, there's flavovirens fluor in the endochylema.
2.Theresult of ELISA:The production and secretion of IL-6,IL-8,TNFαin HUVEC s were significantly induced by LPS treatment(p＜0.01);the elevated level of these cytokines was reduced significantly by simvastatin treated cells when compared with control group(p＜0.01)。
3.The result of real time quantitative PCR:The mRNA expression of IL-6、IL-8、TNFα、uPA/uPAR in HUVECs were significantly induced by LPS treatment(p＜0.05);The mRNA expression of IL-6、IL-8、TNFα、uPA/uPAR in HUVECs reduced significantly by simvastatin treated cells when compared with control group(p＜0.05).
Conclution:
1.The mRNA expression of IL-6、IL-8、TNF-α、uPA/uPAR in HUVECs can be induces by LPS.
2.Simvastatin can reduce the mRNA expression of IL-6、IL-8、TNF-α、uPA/uPAR in the HUVECs stimulaed by LPS.
引文
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[1]Veillard NR,Brannnersreuther V,Arnaud C,et al.Simvastatin modulates chemokine and chemokine receptor expression by geranylgeranyl isoprenoid pathway in human endothelial cells and macrophages[J].Atherosclerosis,2006,188(1):51-58
[2]Mantuano E,Santi S,Filippi C,et al.Simvastatin and fluvastatin reduce interleukin-6 interleukin-8 lipopolysaccharide(LPS)stimulated production by isolated human monocytes from chronic kidney disease patients[J].Biomedicine &Pharmacotherapy,2007,61(6):360-365
[3]Sakoda K,Yamamoto M,Negishi Y,et al.Simvastatin Decreases IL-6 and IL-8 Production in Epithelial Cells[J].J Dent Res,2006,85(6):78-90
[4]吴在德主编《外科学》第五版 人民卫生出版社 2002:185-187
[5]Jaffe EA.Culture of human endothelial cells derived from umbilical veins.Identification by morphologic and immunologic criteria[J].Cell Biol,1973,52(11):2745-56
[6]Jaffe EA,Hoyer LW,Nachman RL.Synthesis of von Willebrand Factor by Cultured Human Endothelial Cells[J].Proc Natl Acad Sci U S A.,1974,71(5):1906-1909
[7]Lan W,Harmon DC,Wang JH,et al.Activated Endothelial Interleukin-1,-6,and -8Concentrations and Intercellular Adhesion Molecule-1Expression Are Attenuated by Lidocaine[J].Anesth Analg,2005,100(2):409-12.
[8]Hooper WC,Phillips DJ,Renshaw MA,et al.The Up-Regulation of IL-6and IL-8 in Human Endothelial Cells by Activated Protein C[J].J Immunol,1998,161(5):2567-73
[9]Wang HR,Li JJ,Huang CX,et al.F1uvastatin inhibits the expression of tumor necrosis factor-alpha and activation of nuclear factor-kappaB in human endothelial cells stimulated by C-reactive protein[J].Clin Chim Acta.2005,353(1-2):53-60
[10]Kawakami A,Tanaka A,Nakajima K,et al.Atorvastatin attenuates remnant lipoprotein induced monocyte adhesion to vascular endothelium under flow conditions[J].Circ Res.2002,91(3):263-71
[11]Leu HB,Chen JW,Wu TC,et al.Effects of fluvastatin,an HMG-CoA reductase inhibitor,on serum levels of interleukin-18 and matrix metallop roteinase-9 in patients with hypercholesterolemia[J].Clin Cardiol 2005,28(9):423-428.