摘要
高血压病(hypertension)发生率高,在中国成人高达18%,目前我国有1.6亿高血压病人。脑卒中(stroke)是高血压病最重要的并发症,中国高血压病人脑卒中的发生率要远远高于欧美国家的高血压病人。中国脑卒中年发病率为2‰,每年新发140万,现有累计700多万患者。脑卒中一旦发生,后果严重,病人往往非死即残。全球每年新发脑卒中1500万,其中500万人死亡,500万人永久性残废。中国每年死于脑卒中的人数约为140万。要使幸存者康复,耗时费力。中国每年因脑卒中直接支出374亿人民币。因此,就脑卒中而言,防止其发生比治疗更重要。如何预防脑卒中的发生?除了降低血压,目前尚无其他有效措施。甚至对于某些病例如缺血性脑卒中,降压治疗也并不能完全阻止脑卒中的发生。因此,有必要对脑卒中的病理生理机制进行深入的研究,找出除了血压以外的更多决定因素,针对这些因素进行处理,提出新的防治策略,从而更好地预防脑卒中的发生,大幅度地降低脑卒中的危害。
脑卒中倾向的自发性高血压大鼠(Stroke-Prone Spontaneously Hypertensive Rats, SHR-SP)是目前研究脑卒中最常用的动物模型,它是从自发性高血压大鼠(Spontaneously Hypertensive Rats, SHR)里筛选出来、定向培育的亚种,具有自发性脑卒中倾向;换言之,并非所有的SHR都会发生脑卒中,SHR与SHR-SP同为高血压动物,但SHR-SP更易发生脑卒中,故采用SHR-SP与普通的SHR进行比较研究,有助于找到除了血压以外的其他决定因子,阐明脑卒中发生的病理机制。
目前,对于某种疾病如脑卒中的研究多数只针对某种或某几种血清或组织中的影响因素,这些因素是基于某种假定成立的预期理论而预先选定的,这些研究的目的也只是为了证实或者推翻这些假定的理论。然而,蛋白质组学(proteomics)这一强大技术手段的出现,打破了上述这些常规手段的限制,可一次检测到成千上万种蛋白质,不局限于任何既定的理论框架,有利于发现新的蛋白质标志物,新的现象,以及新的作用机制。
近年来,蛋白质组学结合生物信息学(bioinformatics)技术手段已经广为应用。而相对于普通的硝酸银染色,考马斯亮蓝染色等方法,双向荧光差异凝胶电泳(two dimensional fluorescent difference gel electrophoresis,2D-DIGE)则更加具有其优越性。2D-DIGE是唯一应用内标(Internal Standard)的蛋白质组学方法,内标是从一项实验中所有样品选取等量蛋白混合而成的,将会出现在每一张胶上,并由配套的分析软件以内标为基准分析所有检测到的蛋白表达差异情况,大大提高了实验的可信度。此外,实验组、对照组以及内标组样品分别由3种荧光标记后混合,同时进行一向和二向分离,最大程度的减少了实验过程中的系统误差,提高了实验的可信度。因此,建立2D-DIGE实验技术平台并应用以比较分析SHR-SP与SHR蛋白质表达、探求脑卒中发生发展新的内在机制正是本课题的主要研究目的。
综上所述,本课题主要拟进行以下几个方面的研究:
2D-DIGE实验技术平台的建立。
采用SHR-SP与普通的SHR进行比较研究,应用2D-DIGE技术分离SHR-SP与SHR的脑组织蛋白,选取差异表达的蛋白进行串联质谱鉴定,并用western blot进行验证。
在上述结果的基础上,深入分析差异蛋白在脑卒中发生中的作用。
具体实验及结果如下:
实验一:2D-DIGE技术平台的建立。以SHR-SP和SHR脑组织蛋白样品为例,以CyDye进行荧光标记,随后进行双向电泳分离蛋白,Typhoon Trio扫描获得荧光图像,配套的DeCyder分析软件对图像进行分析,获得差异蛋白信息。结果:成功的进行了样品的荧光标记测试、荧光标记及双向电泳,并对扫描得到的图像进行了分析。即,成功建立了2D-DIGE实验技术平台。
实验二:SHR-SP与SHR脑组织差异表达蛋白的寻找及验证。利用2D-DIGE实验技术平台,比较分析SHR-SP和SHR脑组织差异蛋白的表达。20周龄雄性SHR-SP和SHR,各5只,提取脑组织蛋白进行双向荧光差异凝胶电泳,获得SHR及SHR-SP脑组织的差异蛋白表达谱。结果发现,在2000多个蛋白点中,找到44个显著性差异表达的蛋白,其中19个在SHR-SP高表达,25个低表达。其中20个蛋白得到基质辅助激光解吸飞行时间串联质谱(MOLDI-TOF/TOF MS)鉴定,在鉴定到的差异表达的蛋白点中,9个在SHR-SP中高表达,11个在SHR-SP中低表达;其中抗氧化蛋白谷胱甘肽巯基转移酶(Glutathione S-transferase,GST)在SHR-SP中显著下调(P<0.05)。Western Blot验证了这一结果,与2D-DIGE是一致的。
实验三:SHR-SP氧化应激水平明显高于SHR。既然抗氧化蛋白在SHR-SP显著下调,我们比较了SHR-SP和SHR的氧化应激水平。20周龄雄性SHR-SP和SHR,各10只,提取脑组织蛋白样品及制备血清样品,比较了两者抗氧化防御系统中总抗氧化能力(total antioxidant capacity,TAC),谷胱甘肽过氧化物酶(glutathione peroxidase,GPx)活性和丙二醛(maleic dialdehyde, MDA)含量。结果发现SHR-SP脑中TAC和GPx均显著低于SHR (12.47±3.02 vs 22.73±3.00, P<0.01; 891.81±108.93 vs 1124.4±160.06, P<0.05),而MDA含量却显著高于SHR(1.11±0.11 vs 0.82±0.11, P<0.05)。血清中也得到了一致的结果,SHR-SP组TAC和GPx显著低于SHR(61.28±15.16 vs 123.6±32.39, P<0.05; 12.14±1.77 vs 18.59±2.42, P<0.05),而MDA含量显著高于SHR(13.33±2.90 vs 5.89±1.21, P<0.05)。
实验四:SHR-SP脑梗死面积明显大于SHR。我们接下来比较了SHR-SP和SHR的脑梗死面积。20周龄雄性SHR-SP和SHR,各15只,电凝法进行大脑中动脉栓塞(middle cerebral artery occlusion, MCAO)手术。术后24 h,行氯化三苯基四氮唑(2,3,5’- Triphenyl Tetrazulium Chloride, TTC)染色,计算机分析系统计算脑梗死面积百分比。结果发现,SHR-SP脑梗死面积显著高于SHR,两者有显著差异(23.0 %±3.3 % vs 31.6 %±5.4 %, P=0.004)。
实验五:维生素C和维生素E合用可以改善SHR-SP的氧化应激水平、减小脑梗死面积。通过以上实验结果,我们推测SHR-SP脑梗死严重可能与其氧化应激损伤较严重有关,本实验对此进行了验证。我们选用最常见的抗氧化剂维生素降低氧化应激水平,减少氧化损伤,再观察脑梗死情况是否减轻。50只16周龄雄性SHR-SP随机分为两组:对照组(n=25)和VCE组(Vitamin E (100mg/kg.d) +Vitamin C (200mg/kg.d), n=25),连续灌胃4周。(1)4周后,检测氧化应激指标TAC、GPx和MDA,每组10只。结果显示,VCE组脑中TAC和GPx活性均显著高于对照组(25.57±6.68 vs 12.47±3.02, P<0.05; 1453.11±126.98 vs 891.81±108.93, P<0.01),MDA含量则显著低于对照组(0.80±0.05 vs 1.11±0.11, P<0.05)。血清中得到了一致的结果,VCE组TAC和GPx活性显著高于对照组(124.75±28.43 vs 61.28±15.16, P<0.05; 19.63±4.84 vs 12.14±1.77, P<0.05),MDA含量则显著低于对照组(7.31±0.86 vs 13.33±2.90, P<0.05)。(2)余下的30只SHR-SP,其中15只连续给维生素4周,对照组给蒸馏水,4周后电凝法行MCAO手术。术后24 h,行TTC染色,计算机分析系统计算脑梗死面积百分比。结果发现,VCE组脑梗死面积显著低于对照组,两者有显著差异(20.8 %±3.8 % vs 32.0 %±4.9 %, P=0.003)。
结论:
成功建立了2D-DIGE实验技术平台,可以有效的进行蛋白质差异表达检测。
应用2D-DIGE技术手段获得了SHR-SP和SHR脑组织的差异表达蛋白列表。其中,抗氧化蛋白在SHR-SP中显著下调。
氧化应激与脑卒中的发生有着密切关系,氧化应激的增强是引起脑卒中的一个重要因素。一方面SHR-SP与SHR氧化应激水平有差异:SHR-SP氧化损伤比SHR严重,同时其抗氧化防御能力又弱于SHR;SHR-SP脑梗死面积明显大于SHR。另一方面,给予抗氧化剂Vitamins提高了SHR-SP的抗氧化能力,并减少其氧化损伤;减少氧化应激可显著减少SHR-SP脑梗死面积。
Although tremendous achievements have been made in the clinical and technological diagnosis of stroke in the past decade, the therapeutic outcome remains unsatisfactory. Stroke remains to be the second commonest cause of death and a leading cause of adult disability worldwide. According to recent estimates published by the World Health Organization, about 15 million people per year fall victim to stroke worldwide, of whom 5 million die and another 5 million are left permanently disabled. Stroke emerges as a condition associated with a combination of multiple risk factors including hypertension, hyperlipidemia and diabetes, of which hypertension is the most prevalent and powerful, across age, sex, and geographic regions.
Individual susceptibility to stroke varies greatly among hypertensive patients. Interestingly, a similar variation in susceptibility to stroke was found in spontaneously hypertensive rats (SHR), and thus a sub-strain of rats was developed based on their susceptibility to stroke from SHR and named stroke-prone spontaneously hypertensive rats (SHR-SP). SHR-SP is a useful and unique animal model for studying the pathogenesis of stroke. These rats were subjected to a stroke at a mean age of 10 months for males and 14 months for females in our laboratory. We speculated that the difference between SHR-SP and SHR might imply some mechanisms underlying the pathogenesis and development of stroke, and that as SHR and SHR-SP are both hypertensive, a comparison of SHR-SP with SHR would provide new insights into the pathogenesis and development of stroke beyond hypertension.
Traditionally, studies on stroke have been performed by one or a few factors in blood plasma/serum or tissues. These factors are usually preselected based on some expected hypothesis, and the aim is to either verify or disprove this hypothesis. However, as a means of unbiased global screening of physiological perturbations, these approaches are limited. This means that detection of unexpected or novel mechanistic phenomena or markers is almost impossible to obtain. For this purpose, a robust method that can simultaneously quantify and identify a large number (hundreds to thousands) of molecules is needed. The integrated multiple systems biology approach has been employed in recent years and has turned out to be an efficient approach for improving our understanding of systems as a whole.
The main aspects of this study are as followings:
1. To establish a hypothesis-free high-throughput strategy capable of resolving several thousands of individual protein spots on a single gel -- two dimensional fluorescent difference gel electrophoresis (2D-DIGE). Each soluble protein sample from SHR-SP and SHR was minimally labeled with 400pmol (1ul) CyDye DIGE fluors dissolved in 99.8% DMF per 50ug of protein (8 nM dye/mg protein), one SHR, SHR-SP and internal standard sample forming a set of Cy2, Cy3, and Cy5 labeled samples was combined and mixed to perform the first dimensional isoelectric focusing (IEF) and second dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We successfully acquired the image of differentially expressed protein profile of the brain of SHR-SP and SHR.
2. To study the differentially expressed protein profile in the brain of SHR-SP and SHR by using 2D-DIGE and matrix assisted laser desorption /ionization- time of flight mass spectrometry (MALDI-TOF MS). Using 2D-DIGE approach, we comparatively analyzed the proteome of SHR-SP and SHR. We reproducibly separated over 2,000 polypeptides using two-dimensional electrophoresis (2DE) at PH range of 3-10. Using DeCyder software to process the 2D gel images, forty-five protein spots were differentially expressed in the brains of the two kinds of animals. Of these, nineteen highly expressed in SHR-SP, and twenty-five highly expressed in SHR. Twenty proteins were identified with high confidence by MALDI-TOF/TOF MS. Our data suggested that some anti-oxidant proteins including glutathione S-transferase (GST) pi2 and GST A5 were down-regulated in the brain of SHR-SP.
3. Since the anti-oxidant proteins were down-regulated in SHR-SP when compared with SHR, oxidative stress indicators including total anti-oxidant capacity (TAC), glutathione peroxidase (GPx) activity and maleic dialdehyde (MDA) were measured (n=10 in each group). The results showed that TAC level and GPx activity in brain of SHR-SP were significantly lower than those in SHR (12.47±3.02 vs 22.73±3.00, P<0.01; 891.81±108.93 vs 1124.4±160.06, P<0.05), while MDA level in SHR-SP was significantly higher than that in SHR (1.11±0.11 vs 0.82±0.11, P<0.05). The results were the same in the serum. The TAC level and GPx activity in SHR-SP were significantly lower than those in SHR (61.28±15.16 vs 123.6±32.39, P<0.05; 12.14±1.77 vs 18.59±2.42, P<0.05), while MDA level was significantly higher than that in SHR (13.33±2.90 vs 5.89 ±1.21, P<0.05).
4. The above results revealed that oxidative damage was more severe in SHR-SP than that in SHR. Furthermore, we investigated the pathological importance of oxidative stress in the pathogenesis and development of stroke in SHR-SP. Twenty male SHR-SP and SHR aged 5 months were subjected to middle cerebral artery occlusion (MCAO) and the infarct area was measured (n=10 in each group). TTC staining showed that the infarct area in SHR-SP was lager than that in SHR. Using the computerized image analysis system, the mean infarct area of SHR-SP and SHR was 31.6 %±5.4 % and 23.0 %±3.3 %, respectively. Unpaired t-test showed that the mean infarct area of SHR-SP was significantly larger than that of SHR (P=0.004).
5. The infarct area of SHR-SP in our work was significantly larger than that of SHR, suggesting that the increase in the cerebral ischemia infarct area may be associated with a higher level of oxidative stress in SHR-SP. In order to confirm the association between high oxidative stress and increased infarct area of SHR-SP, the animals were treated with vitamins C and E to improve the antioxidant defense system. Fifty male SHR-SP were randomly divided into control group (n=25) and vitamins treatment group (vitamin E and C (100 + 200 mg / kg. d) by gavage for 4 weeks, n=25). After 4-week treatment, twenty SHR-SP were killed for detection of TAC, GPx and MDA (n=10 in each group). The results showed that TAC level and GPx activity in brain of vitamin treated group were significantly higher than those in control (25.57±6.68 vs 12.47±3.02, P<0.05; 1453.11±126.98 vs 891.81±108.93, P<0.01), while MDA level in vitamin treated group was significantly lower than that in control (0.80±0.05 vs 1.11±0.11, P<0.05). The results were the same in the serum. The TAC level and GPx activity in vitamin treated group were significantly higher than those in control (124.75±28.43 vs 61.28±15.16, P<0.05; 19.63±4.84 vs 12.14±1.77, P<0.05), while MDA level was significantly lower than that in control (7.31±0.86 vs 13.33±2.90, P<0.05). The remaining thirty SHR-SP underwent MCAO and the infarct area was measured. TTC staining showed that the infarct area of SHR-SP control and vitamin treated group was 32.0 %±4.9 % and 20.8 %±3.8 %, respectively. Unpaired t-test showed that the infarct area of VCE group was significantly smaller than that of the control group (P=0.003).
In conclusion:
1. We successfully established the technology of 2D-DIGE and with 2D-DIGE we can detect the differentially expressed protein successfully.
2. With 2D-DIGE, we acquired the differentially expressed protein profiles of SHR-SP and SHR. Of the multitudinous proteins, we detected a significant decrease in anti-oxidative proteins (GST Pi 2, and GST A5) of SHR-SP when compared with SHR.
3. Oxidative stress plays an important role in the pathogenesis and development of cerebral ischemia: On the one hand, oxidative damage was more severe in SHR-SP than that in SHR, and the infarct area after MCAO was significantly larger in SHR-SP than that in SHR. On the other hand, vitamins significantly decreased the oxidative stress level of SHR-SP, and the decreased oxidative stress resulted in a decrease in the infarct area in SHR-SP.
引文
1. Macri J and Rapundalo ST. Application of proteomics to the study of cardiovascular biology. Trends Cardiovasc Med 2001; 11: 66-75
2. Dunn MJ. Studying heart disease using the proteomic approach. Drug Discov Today 2000; 5: 76-84
3. Banks RE, Dunn MJ, Hochstrasser DF, Sanchez JC, Blackstock W, Pappin DJ, and Selby PJ. Proteomics: new perspectives, new biomedical opportunities. Lancet 2000; 356: 1749-1756
4. McGregor E and Dunn MJ. Proteomics of heart disease. Human Molecular Genetics 2003; 12: R135-R144
5. Arrell DK, Neverova I, and Van Eyk JE. Cardiovascular Proteomics: Evolution and Potential. Circ. Res. 2001; 88: 763– 773
6. Weeks ME, Sinclair J, Butt A, Chung YL, Worthington JL, Wilkinson CR, Griffiths J, Jones N, Waterfield MD, and Timms JF. A parallel proteomic and metabolomic analysis of the hydrogen peroxide- and Sty1p-dependent stress response in Schizosaccharomyces pombe. Proteomics 2006, 6, 2772-2796
7. Craig A, Sidaway J, Holmes E, Orton T, Jackson D, Rowlinson R, Nickson J, Tonge R, Wilson I, and Nicholson J. Systems toxicology: integrated genomic, proteomic and metabonomic analysis of methapyrilene induced hepatotoxicity in the rat. J Proteome Res 2006, 5, 1586-1601
8. Alban A, David SO, Bjorkesten L, Andersson C, Sloge E, Lewis S, and Currie I. A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 2003, 3, 36 - 44
9. Bredemeyer AJ, Lewis RM, Malone JP, Davis AE, Gross J, Townsend RR, and Ley TJ. MEDICAL SCIENCES: A proteomic approach for the discovery of protease substrates. PNAS 2004, 101, 11785 - 11790
10. Tang WQ, Deng ZP, Oses-Prieto A, Suzuki N, Zhu SW, Zhang X, Burlingame AL, and Wang ZY. Proteomics Studies of Brassinosteroid Signal Transduction Using Prefractionation and Two-dimensional DIGE. Mol. Cell. Proteomics 2008; 7: 728 - 738
11. Crecelius A, Gotz A, Arzberger T, Frohlich T, Arnold GJ, Ferrer I, and Kretzschmar HA. Assessing quantitative post-mortem changes in the gray matter of the humanfrontal cortex proteome by 2-D DIGE. Proteomics 2008; 8: 1276-91
12. Karp NA and Lilley KS. Investigating sample pooling strategies for DIGE experiments to address biological variability. Proteomics 2009; 9: 388-97
13. Friedman DB, Hill S, Keller JW, Merchant NB, Levy SE, Coffey RJ, and Caprioli RM. Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics 2004; 4: 793-811
1. Caplan LR. Treatment of acute stroke: still struggling. JAMA 2004; 292: 1883 - 1885.
2. Liu M, Wu B, Wang WZ, Lee LM, Zhang SH, and Kong LZ. Stroke in China: epidemiology, prevention, and management strategies. Lancet Neurol 2007; 6: 456 - 464.
3. Bonita R, Mendis S, Truelsen T, Bogousslavsky J, Toole J, and Yatsu F. The Global Stroke Initiative. Lancet Neurol 2004; 3: 391 - 393.
4. Mackay J and Mensah G. Global burden of stroke, in The Atlas of Heart Disease and Stroke. Geneva, Switzerland: World Health Organization 2004
5. Cubrilo-Turek M. Stroke risk factors: recent evidence and new aspects. International Congress Series 2004; 1262: 466 - 469.
6. Fang XH, Zhang XH, Yang QD, Dai XY, Su FZ, Rao ML, Wu SP, Du XL, Wang WZ, and Li SC. Subtype Hypertension and Risk of Stroke in Middle-Aged and Older Chinese: A 10-Year Follow-Up Study. Stroke 2006; 37: 38 - 43.
7. Zhang XF, Attia J, D’Este C, and Yu XH. Prevalence and Magnitude of Classical Risk Factors for Stroke in a Cohort of 5092 Chinese Steelworkers Over 13.5 Years of Follow-up. Stroke 2004; 35: 1052 - 1056.
8. Rietbrock S, Heeley E, Plumb J, and Staa T. Chronic atrial fibrillation: Incidence, prevalence, and prediction of stroke using the Congestive heart failure, Hypertension, Age >75, Diabetes mellitus, and prior Stroke or transient ischemic attack (CHADS2) risk stratification scheme. Am Heart J 2008; 156: 57 - 64.
9. Olsen TS, Christensen RHB, Kammersgaard LP, and Andersen KK. Higher Total Serum Cholesterol Levels Are Associated With Less Severe Strokes and Lower All-Cause Mortality: Ten-Year Follow-Up of Ischemic Strokes in the Copenhagen Stroke Study. Stroke 2007; 38: 2646 - 2651.
10. Okamoto K, Yamori Y, and Nagaoka A. Establishment of the stroke-prone spontaneously hypertensive rats (SHR). Circ Res 1974; 34: I-143 - I-145
11. Zhang W, Liu AJ, Yi-Ming W, Liu JG, Shen FM, and Su DF. Pressor and non-pressor effects of sodium loading on stroke in stroke-prone spontaneously hypertensive rats. Clin Exp Pharmacol Physiol 2008; 35: 83 - 88.
12. Liu AJ, Ma XJ, Shen FM, Liu JG, Chen H, and Su DF. Arterial Baroreflex. A Novel Target for Preventing Stroke in Rat Hypertension. Stroke 2007; 38: 1916 - 1923.
13. Bredemeyer AJ, Lewis RM, Malone JP, Davis AE, Gross J, Townsend RR, and Ley TJ.MEDICAL SCIENCES: A proteomic approach for the discovery of protease substrates. PNAS 2004; 101: 11785 - 11790
14. Tang WQ, Deng ZP, Oses-Prieto A, Suzuki N, Zhu SW, Zhang X, Burlingame AL, and Wang ZY. Proteomics Studies of Brassinosteroid Signal Transduction Using Prefractionation and Two-dimensional DIGE. Mol. Cell. Proteomics 2008; 7: 728 - 738
15. Fang XH, Zhang XH, Yang QD, Dai XY, Su FZ, Rao ML, Wu SP, Du XL, Wang WZ, and Li SC. Subtype Hypertension and Risk of Stroke in Middle-Aged and Older Chinese: A 10-Year Follow-Up Study. Stroke 2006; 37: 38 - 43.
1. Chen K, Thomas SR, and Keaney JF. Beyond LDL oxidation: ROS in vascular signal transduction. Free Radic Biol Med 2003; 35: 117 - 132
2. Stocker R and Keaney JF. Role of oxidative modifications in atherosclerosis. Physiol Rev 2004; 84: 1381- 1478
3. Griendling KK and Fitzgerald GA. Oxidative stress and cardiovascular injury. Animal and human studies. Circulation 2003;108:2034 - 2040
4. Madamanchi NR, Vendrov A, and Runge MS. Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol 2005; 25: 29 - 38
5. Mueller CFH, Laude K, McNally JS, and Harrison DG. Redox mechanisms in blood vessels. Arterioscler Thromb Vasc Biol 2005; 25: 274 - 278
6. Okamoto K, Yamori Y, and Nagaoka A. Establishment of the stroke-prone spontaneously hypertensive rats (SHR). Circ Res 1974; 34: I-143 - I-145
7. Zhang W, Liu AJ, Yi-Ming W, Liu JG, Shen FM, and Su DF. Pressor and non-pressor effects of sodium loading on stroke in stroke-prone spontaneously hypertensive rats. Clin Exp Pharmacol Physiol 2008; 35: 83 - 88.
8. Liu AJ, Ma XJ, Shen FM, Liu JG, Chen H, and Su DF. Arterial Baroreflex. A Novel Target for Preventing Stroke in Rat Hypertension. Stroke 2007; 38: 1916 - 1923.
9. Erel O. A novel automated method to measure total antioxidant response against potent free radical reactions. Clin Biochem 2004; 37: 112 - 119.
10. Schuessel K, Schafer S, Bayer TA, Czech C, Pradier L, Muller-Spahn F, Muller WE, and Eckert A. Impaired Cu/Zn-SOD activity contributes to increased oxidative damage in APP transgenic mice. Neurobiol Dis 2005; 18: 89 - 99.
11. Mihara M and Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1978; 86: 271 - 278.
12. Tamura A, Graham DI, McCulloch J, and Teasdale GM. Focal cerebral ischemia in the rat: Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J Cereb Blood Flow Metab 1981; 1: p53 - p60.
13. Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, and Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 1986; 17: 472 - 476.
14. Joshi CN, Jain SK, and Murthy PS. An optimized triphenyltetrazolium chloride method for identification of cerebral infarcts. Brain Res Brain Res Protoc 2004; 13:11-17
15. Alexandrova ML and Bochev PG.Oxidative stress during the chronic phase after stroke. Free Radic Biol Med 2005; 39: 297-316.
16. Yu Y, Fukuda N, Yao EH, Matsumoto T, Kobayashi N, Suzuki R, Tahira Y, Ueno T, and Matsumoto K. Effects of an ARB on endothelial progenitor cell function and cardiovascular oxidation in hypertension. Am J Hypertens 2008; 21: 72 - 77.
17. Moro MA, Almeida A, Bolanos JP, and Lizasoain I. Mitochondrial respiratory chain and free radical generation in stroke. Free Radic Biol Med 2005; 39: 1291 - 1304.
18. El-Kossi MM and Zakhary MM. Oxidative stress in the context of acute cerebrovascular stroke. Stroke 2000; 31: 1889 - 1892.
19. Yao EH, Fukuda N, Matsumoto T, Kobayashi N, Katakawa M, Yamamoto C, Tsunemi A, Suzuki R, Ueno T, and Matsumoto K. Losartan improves the impaired function of endothelial progenitor cells in hypertension via an antioxidant effect. Hypertens Res 2007; 30: 1119 - 1128.
20. Nagotani S, Hayashi T, Sato K, Zhang W, Deguchi K, Nagano I, Shoji M, and Abe K. Reduction of Cerebral Infarction in Stroke-Prone Spontaneously Hypertensive Rats by Statins Associated With Amelioration of Oxidative Stress. Stroke 2005; 36: 670 - 672.
21. McBride MW, Brosnan MJ, Mathers J, McLellan LI, Miller WH, Graham D, Hanlon N, Hamilton CA, Polke JM, Lee WK, and Dominiczak AF. Reduction of Gstm1 Expression in the Stroke-Prone Spontaneously Hypertension Rat Contributes to Increased Oxidative Stress. Hypertension 2005; 45: 786 - 792.
22. Warner DS, Sheng HX, and Batinic-Haberle I. Oxidants, antioxidants and the ischemic brain. J Exp Biol 2004; 207: 3221 - 3231.
23. Cherubini A, Ruggiero C, Polidori MC, and Mecocci P. Potential markers of oxidative stress in stroke. Free Radic Biol Med 2005; 39: 841 - 852.
24. Cui X, Zuo P, Zhang Q, Li X, Hu Y, Long J, Packer L, and Liu J. Chronic systemic D-galactose exposure induces memory loss, neurodegeneration, and oxidative damage in mice: protective effects of R-alpha-lipoic acid. J Neurosci Res 2006; 84: 647-654.
25. Serarslan G, Altug E, Kontas T, Atik E, and Avci G. Caffeic acid phenethyl ester accelerates cutaneous wound healing in a rat model and decreases oxidative stress. Clin Exp Dermatol 2007; 32: 709 - 715.
26. Balog T, Sobocanec S, Sverko V, Krolo I, Rocic B, Marotti M, and Marotti T.The influence of season on oxidant-antioxidant status in trained and sedentary subjects.Life Sci 2006; 78: 1441 - 1447.
27. Voetsch B, Jin RC, Bierl C, Benke KS, Kenet G, Simioni P, Ottaviano F, Damasceno BP, Annichino-Bizacchi JM, Handy DE, and Loscalzo J. Promoter Polymorphisms in the Plasma Glutathione Peroxidase (GPx-3) Gene: A Novel Risk Factor for Arterial Ischemic Stroke Among Young Adults and Children. Stroke 2007; 38: 41 - 49.
28. Voetsch B, Jin RC, Bierl C, Deus-Silva L, Camargo ECS, Annichino-Bizacchi JM, Handy DE, and Loscalzo J. Role of Promoter Polymorphisms in the Plasma Glutathione Peroxidase (GPx-3) Gene as a Risk Factor for Cerebral Venous Thrombosis. Stroke 2008; 39: 303 - 307.
29. Pialoux V, Foster GE, Brugniaux JV, Duggan CT, Hanly PJ, and Poulin MJ. Effect of 4 days of intermittent hypoxia on oxidative stress in healthy men. FASEB J 2008; 22: 960 - 963.
30. Prasad K. Hypocholesterolemic and antiatherosclerotic effect of flax lignan complex isolated from flaxseed. Atherosclerosis 2005; 179: 269 - 275.
31. Polidori MC, Frei B, Cherubini A, Nelles G, Rordorf G, Keaney JF, Schwamm L, Mecocci P, Koroshetz WJ, and Beal MF. Increased plasma levels of lipid hydroperoxides in patients with ischemic stroke. Free Radic Biol Med 1998; 25: 561 - 567.
1. Corre^a MC, Maldonado P, Rosa CS, Lunkes G, Lunkes DS, Kaizer RR, Ahmed M, Morsch VM, Pereira ME, and Schetinger MR. Oxidative stress and erythrocyte acetylcholinesterase (AChE) in hypertensive and ischemic patients of both acute and chronic stages. Biomedicine & Pharmacotherapy 2008; 62: 317 - 324
2. Lagowska-Lenard M, Bielewicz J, Raszewski G, Stelmasiak Z, and Bartosik-Psujek H. Oxidative stress in cerebral stroke. Pol Merkur Lekarski 2008; 25: 205-208.
3. Alexandrova ML and Bochev PG. Oxidative stress during the chronic phase after stroke. Free Radic Biol Med 2006; 39: 297 - 316.
4. Moro MA, Almeida A, Bolanos JP, and Lizasoain, I. Mitochondrial respiratory chain and free radical generation in stroke. Free Radic Biol Med 2005; 39: 1291 - 1304.
5. McCluskey S, Hall M, Stanton C, and Devery R. Alphatocopherol inhibits oxidative stress induced by cholestanetriol and 25-hydroxycholesterol in porcine ovarian granulosa cells. Mol. Cell. Biochem. 1999; 194: 217 - 225.
6. Ferretti G, Baccheni T, and Masciangelo S. Lipid peroxidation in hemodialysis patients: effect of vitamin C supplementation. Clin Biochem 2008; 41: 381 - 386.
7. Noguchi T, Ikeda K, Sasaki Y, Yamamoto J, and Yamori Y. Effects of vitamin E and sesamin on hypertension and cerebral thrombogenesis in stroke-prone spontaneously hypertensive rats. Clin Exp Pharmacol Physiol 2004; 31: S24 - S26.
8. Chen X, Touyz RM, Park JB, and Schiffrin EL. Antioxidant Effects of Vitamins C and E Are Associated With Altered Activation of Vascular NADPH Oxidase and Superoxide Dismutase in Stroke-Prone SHR. Hypertension 2001; 38: 606 - 611
9. Patra RC, Swarup D, and Dwivedi SK. Antioxidant effects ofα-tocopherol, ascorbic acid and L-methionine on lead induced oxidative stress to the liver, kidney and brain in rats. Toxicology 2001; 162: 81 - 88
10. Kamada H, Yu F, Nito C, and Chan PH. Influence of Hyperglycemia on Oxidative Stress and Matrix Metalloproteinase-9 Activation After Focal CerebralIschemia/Reperfusion in Rats. Stroke 2007; 38: 1044 - 1049
11. Liu KJ and Rosenberg GA. Matrix metalloproteinases and free radicals in cerebral ischemia. Free Radic Biol Med 2005; 39: 71 - 80.
12. Poulet R, Gentile MT, Vecchione C, Distaso M, Aretini A, Fratta L, Russo G, Echart C, Maffei A, De Simoni MG, and Lembo G. Acute hypertension induces oxidative stress in brain tissues. J Cereb Blood Flow Metab 2006; 26: 253 - 262.
13. Chan PH. Reactive oxygen radicals in signaling and damage in the ischemic brain. J Cereb Blood Flow Metab 2001; 21: 2 - 14.
14. Huang JY, Liao JW, Liu YC, Lu SY, Chou CP, Chan WH, Chen SU, and Ueng TH. Motorcycle Exhaust Induces Reproductive Toxicity and Testicular Interleukin-6 in Male Rats. Toxicological Sciences 2008; 103:137 - 148.
15. Ramanathan K, Balakumar BS, and Panneerselvam C.Effects of ascorbic acid and a-tocopherol on arsenic-induced oxidative stress. Human & Experimental Toxicology 2002; 21: 675 - 680
16. Gomez-Lazaro M, Galindo MF, Melero-Fernandez de Mera RM, Fernandez-Gómez FJ, Concannon CJ, Segura MF, Comella GX, Prehn JHM, and Jordan J. Reactive Oxygen Species and p38 Mitogen-Activated Protein Kinase Activate Bax to Induce Mitochondrial Cytochrome c Release and Apoptosis in Response to Malonate. Mol Pharmacol 2007; 71: 736 - 743.
17. Chapple1 ILC, Milward1 MR, and Dietrich T.The Prevalence of Inflammatory Periodontitis Is Negatively Associated with Serum Antioxidant Concentrations. J. Nutr 2007; 137: 657 - 664.
18. Milman U, Blum S, Shapira C, Aronson D, Miller-Lotan R, Anbinder Y, Alshiek J, Bennett L, Kostenko M, Landau M, Keidar S, Levy Y, Khemlin A, Radan A, and Levy AP. Vitamin E Supplementation Reduces Cardiovascular Events in a Subgroup of Middle-Aged Individuals With Both Type 2 Diabetes Mellitus and the Haptoglobin 2-2 Genotype. Arteriosclerosis, Thrombosis, and Vascular Biology 2008; 28: 341 - 347
1. Macri J and Rapundalo ST. Application of proteomics to the study of cardiovascular biology. Trends Cardiovasc Med 2001; 11: 66 - 75
2. Dunn MJ. Studying heart disease using the proteomic approach. Drug Discov Today 2000; 5: 76 - 84
3. Banks RE, Dunn MJ, Hochstrasser DF, Sanchez JC, Blackstock W, Pappin DJ, and Selby PJ. Proteomics: new perspectives, new biomedical opportunities. Lancet 2000; 356: 1749 - 1756
4. McGregor E and Dunn MJ. Proteomics of heart disease. Human Molecular Genetics 2003; 12: R135 - R144
5. Arrell DK, Neverova I, and Eyk JEV. Cardiovascular Proteomics: Evolution and Potential. Circ. Res. 2001; 88: 763– 773
6. Wasinger VC, Cordwell SJ, Cerpa-Poljak A, Yan JX, Gooley AA, Wilkins MR, Duncan MW, Harris R, Williams KL, and Humphery-Smith I. Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis 1995; 16: 1090 - 1094
7. Anderson NL and Anderson NG. Proteome and proteomics: new technologies, new concepts, and new words. Electrophoresis 1998; 19: 1853 - 1861
8. Jager D, Jungblut PR, and Muller-Werdan U. Separation and identification of human heart proteins. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 771: 131 - 153
9. O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 1975; 250: 4007 - 4021
10. Marshall T and Williams KM. Proteomics and its impact upon biomedical science. Br J Biomed Sci 2002; 59: 47 - 64
11. Hoving S, Voshol H, and van Oostrum J. Towards high performance two-dimensional gel electrophoresis using ultrazoom gels. Electrophoresis 2000; 21: 2617 - 2621
12. Wildgruber R, Harder A, Obermaier C, Boguth G, Weiss W, Fey SJ, Larsen PM, and Gorg A. Towards higher resolution: two-dimensional electrophoresis of Saccharomyces cerevisiae proteins using overlapping narrow immobilized pH gradients. Electrophoresis 2000; 21: 2610– 2616
13. Westbrook JA, Wheeler JX, Wait R, Welson SY, and Dunn MJ. The human heart proteome: Two-dimensional maps using narrow-range immobilised pH gradients.Electrophoresis 2006; 27: 1547– 1555
14. Yates 3rd JR. Mass spectrometry. From genomics to proteomics. Trends Genet 2000; 16: 5 - 8
15. PK Jensen, L Pasa-Tolic, KK Peden, Martinovic S, Lipton MS, Anderson GA, Tolic N, Wong KK, and Smith RD. Mass spectrometric detection for capillary isoelectric focusing separations of complex protein mixtures. Electrophoresis 2000; 21: 1372 - 1380
16. Bredemeyer AJ, Lewis RM, Malone JP, Davis AE, Gross J, Townsend RR, and Ley TJ. A proteomic approach for the discovery of protease substrates. PNAS 2004; 101: 11785 - 11790
17. Lanne B and Panfilov O. Protein staining influences the quality of mass spectra obtained by peptide mass fingerprinting after separation on 2-d gels. A comparison of staining with coomassie brilliant blue and sypro ruby. J Proteome Res 2005; 4: 175 - 179
18. Unlu M, Morgan ME, and Minden JS. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 1997; 18: 2071 - 2077
19. Corthals GL, Gygi SP, Aebesold R, and Patterson SD. Identification of proteins by mass spectrometry. Rabilloud T. Proteome research: two-dimensional gel electrophoresis and identification methods. Springer, Berlin, Heidelberg, New York 2000, pp. 197 - 231.
20. Andersen JS and Mann M. Functional genomics by mass spectrometry. FEBS Lett 2000; 480: 25 - 31
21. Hortin GL. The MALDI-TOF Mass Spectrometric View of the Plasma Proteome and Peptidome. Clin. Chem. 2006; 52: 1223 - 1237
22. Blueggel M, Chamrad D, and Meyer HE. Bioinformatics in proteomics. Curr Pharm Biotechnol 2004; 5: 79 - 88
23. Lemkin PF. Comparing 2-D electrophoretic gels across Internet databases. Methods Mol Biol 1999; 112: 393 - 410
24. Edwards AVG, White MY, and Cordwell SJ. Mol. Cell. The role of proteomics in clinical cardiovascular biomarker discovery. Proteomics 2008; 10.1074/mcp.R800007 - MCP200
25. Huang ZY, Yang PY, Almofti MR, Yu YL, Rui YC, and Yang PY. Comparativeanalysis of the proteome of left ventricular heart of arteriosclerosis in rat. Life Sci 2004; 75: 3103 - 3115
26.顾劲扬。心肌缺血预适应的研究进展。中国康复医学杂志,2005,20(2): 153 - 155
27. Schwertz H, Langin T, Platsch H, Richert J, Bomm S, Schmidt M, Hillen H, Blaschke G, Meyer J, Darius H, and Buerke M. Two-dimensional analysis of myocardial protein expression following myocardial ischemia and reperfusion in rabbits. Proteomics 2002; 2: 988– 995
28. Kim N, Lee Y, Kim H, Joo H, Youm JB, Park WS, Warda M, Cuong DV, and Han J. Potential biomarkers for ischemic heart damage identified in mitochondrial proteins by comparative proteomics. Proteomics 2006; 6: 1237 - 1249
29. White MY, Cordwell SJ, McCarron HC, Prasan AM, Craft G, Hambly BD, and Jeremy RW. Proteomics of ischemia/reperfusion injury in rabbit myocardium reveals alterations to proteins of essential functional systems. Proteomics 2005; 5: 1395 - 1410
30. Sawicki G and Jugdutt BI. Detection of regional changes in protein levels in the in vivo canine model of acute heart failure following ischemia-reperfusion injury: functional proteomics studies. Proteomics 2004; 4: 2195 - 2202
31. Jugdutt BI and Sawicki G. AT1 receptor blockade alters metabolic, functional and structural proteins after reperfused myocardial infarction: detection using proteomics. Mol Cell Biochem 2004; 263: 179 - 188
32. Chu GX, Egnaczyk GF, Zhao W, Jo SH, Fan GC, Maggio JE, Xiao RP, and Kranias EG. Phosphoproteome Analysis of Cardiomyocytes Subjected to ?-Adrenergic Stimulation: Identification and Characterization of a Cardiac Heat Shock Protein p20. Circ. Res. 2004; 94: 184 - 193
33. Marshall J, Kupchak P, Zhu W, Yantha J, Vrees T, Furesz S, Jacks K, Smith C, Kireeva I, Zhang R, Takahashi M, Stanton E, and Jackowski G. Processing of serum proteins underlies the mass spectral fingerprinting of myocardial infarction. J Proteome Res 2003; 2: 361 - 372
34. Mateos-Cáceres PJ, García-Méndez A, FarréAL, Macaya C, Nú?ez A, Gómez J, Alonso-Orgaz S, Carrasco C, Burgos ME, de Andrés R, Granizo JJ, FarréJ, and Rico LA. Proteomic analysis of plasma from patients during an acute coronary syndrome. J. Am. Coll. Cardiol. 2004; 44: 1578 - 1583
35. Corbett JM, Why HJ, Wheeler CH, Richardson PJ, Archard LC, Yacoub MH, andDunn MJ. Cardiac protein abnormalities in dilated cardiomyopathy detected by two-dimensional polyacrylamide gel electrophoresis. Electrophoresis 1998; 19: 2031 - 2042
36. Weekes J, Wheeler CH, Yan JX, Weil J, Eschenhagen T, Scholtysik G, and Dunn MJ. Bovine dilated cardiomyopathy: proteomic analysis of an animal model of human dilated cardiomyopathy. Electrophoresis 1999; 20: 898 - 906
37. Weekes J, Morrison K, Mullen A, Wait R, Barton P, and Dunn MJ. Hyperubiquitination of proteins in dilated cardiomyopathy. Proteomics 2003; 3: 208 - 216
38. Buscemi N, Doherty-Kirby A, Sussman MA, Lajoie G, and Van Eyk JE. Proteomic analysis of Rac1 transgenic mice displaying dilated cardiomyopathy reveals an increase in creatine kinase M-chain protein abundance. Mol Cell Biochem 2003; 251: 145 - 151
39. Heinke MY, Wheeler CH, Chang D, Einstein R, Drake-Holland A, Dunn MJ, and dos Remedios CG. Protein changes observed in pacing-induced heart failure using two-dimensional electrophoresis. Electrophoresis 1998; 19: 2021 - 2030
40. Haghighi K, Gregory KN, and Kranias EG. Sarcoplasmic reticulum Ca-ATPase-phospholamban interactions and dilated cardiomyopathy. Biochem Biophys Res Commun 2004; 322: 1214 - 1222
41. Chu GX, Kerr JP, Mitton B, Egnaczyk GF, Vazquez JA, Shen M, Kilby GW, Stevenson TI, Maggio JE, Vockley J, Rapundalo ST, and Kranias EG. Proteomic analysis of hyperdynamic mouse hearts with enhanced sarcoplasmic reticulum calcium cycling. FASEB J 2004; 18: 1725 - 1727
42. Arnott D, O'Connell KL, King KL, and Stults JT. An Integrated Approach to Proteome Analysis: Identification of Proteins Associated with Cardiac Hypertrophy. Analytical Biochemistry 1998; 258: 1 - 18
43. Jiang L, Tsubakihara M, Heinke MY, Yao M, Dunn MJ, Phillips W, dos Remedios CG, and Nosworthy NJ. Heart failure and apoptosis: electrophoretic methods support data from micro- and macro-arrays. A critical review of genomics and proteomics. Proteomics 2001; 1: 1481 - 1488
44.张其鹏,张丹,刘贝,朱晓华,卢铭,陈光慧,尚彤,汤健。心力衰竭相关基因和蛋白质数据库的初构。北京大学学报(医学版),2002,34:276 - 280
45. Yan L, Ge H, Li H, Lieber SC, Natividad F, Resuello RR, Kim SJ, Akeju S, Sun A,Loo K, Peppas AP, Rossi F, Lewandowski ED, Thomas AP, Vatner SF, and Vatner DE. Gender-specific proteomic alterations in glycolytic and mitochondrial pathways in aging monkey hearts. J Mol Cell Cardiol 2004; 37: 921 - 929
46. Kanski J, Behring A, Pelling J, and Sch?neich C. Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effects of biological aging. Am J Physiol Heart Circ Physiol 2005; 288: H371 - H381
47. Liu XH, Qian LJ, Gong JB, Shen J, Zhang XM, and Qian XH. Proteomic analysis of mitochondrial proteins in cardiomyocytes from chronic stressed rat. Proteomics 2004; 4: 3167 - 3176
48. Gibbons GH, Liew CC, Goodarzi MO, Rotter JI, Hsueh WA, Siragy HM, Pratt R, and Dzau VJ. Genetic Markers: Progress and Potential for Cardiovascular Disease. Circulation 2004; 109: IV-47 - IV-58
49. Pinet F, Poirier F, Fuchs S, Tharaux PL, Caron M, Corvol P, Michel JB, and Joubert-Caron R. Troponin T as a marker of differentiation revealed by proteomic analysis in renal arterioles. FASEB J 2004; 18: 585 - 586
50. Thongboonkerd V, Gozal E, Sachleben LR, Arthur JM, Pierce WM, Cai J, Chao J, Bader M, Pesquero JB, Gozal D, and Klein JB. Proteomic Analysis Reveals Alterations in the Renal Kallikrein Pathway during Hypoxia-Induced Hypertension. J. Biol. Chem. 2002; 277: 34708 - 34716
51. Nomoto K, Oguchi S, Watanabe I, Kushiro T, and Kanmatsuse K. Involvement of inflammation in acute coronary syndromes assessed by levels of high-sensitivity C-reactive protein, matrix metalloproteinase-9 and soluble vascular-cell adhesion molecule-1. J Cardiol 2003; 42: 201 - 206
52. Blankenberg S, Barbaux S, and Tiret L. Adhesion molecules and atherosclerosis. Atherosclerosis 2003; 170: 191 - 203
53. Duran MC, Mas S, Martin-Ventura JL, Meilhac O, Michel JB, Gallego-Delgado J, Lazaro A, Tunon J, Egido J, and Vivanco F. Proteomic analysis of human vessels: application to atherosclerotic plaques. Proteomics 2003; 3: 973 - 978
54. You SA, Archacki SR, Angheloiu G, Moravec CS, Rao S, Kinter M, Topol EJ, and Wang Q. Proteomic approach to coronary atherosclerosis shows ferritin light chain as a significant marker: evidence consistent with iron hypothesis in atherosclerosis. Physiol Genomics 2003; 13: 25 - 30
55. Kaul D, Kaur R, Baba I, and Singh D. Functional proteomics of receptor-Ck in thedevelopmental stages of human atherosclerotic arterial wall. Indian Heart J 2003; 55: 252 - 255
56. Mayr M, Siow R, Chung YL, Mayr U, Griffiths JR, and Xu Q. Proteomic and Metabolomic Analysis of Vascular Smooth Muscle Cells Role of PKCδ. Circ Res 2004; 94: e87 - e96.
57. Khanna A. Concerted effect of transforming growth factor-β, cyclin inhibitor p21, and c-myc on smooth muscle cell proliferation. Am J Physiol Heart Circ Physiol 2004; 286: H1133 - H1140
58. Taurin S, Seyrantepe V, Orlov SN, Tremblay TL, Thibault P, Bennett MR, Hamet P, and Pshezhetsky AV. Proteome Analysis and Functional Expression Identify Mortalin as an Antiapoptotic Gene Induced by Elevation of [Na+]i/[K+]i Ratio in Cultured Vascular Smooth Muscle Cells. Circ. Res. 2002; 91: 915 - 922
59. Bruneel A, Labas V, Mailloux A, Sharma S, Vinh J, Vaubourdolle M, and Baudin B. Proteomic study of human umbilical vein endothelial cells in culture. Proteomics 2003; 3: 714 - 723
60. Kamino H, Hiratsuka M, Toda T, Nishigaki R, Osaki M, Ito H, Inoue T, and Oshimura M.. Searching for genes involved in arteriosclerosis: proteomic analysis of cultured human umbilical vein endothelial cells undergoing replicative senescence. Cell Struct Funct 2003; 28: 495 - 503
61. Sprenger RR, Speijer D, Back JW, De Koster CG, Pannekoek H, and Horrevoets AJ. Comparative proteomics of human endothelial cell caveolae and rafts using two-dimensional gel electrophoresis and mass spectrometry. Electrophoresis 2004; 25: 156 - 172
62. Yu M, Chen DM, Hu G, and Wang H. Proteomic response analysis of endothelial cells of human coronary artery to stimulation with carbachol. Acta Pharmacol Sin 2004; 25: 1124 - 1130
63. Muth E, Driscoll WJ, Smalstig A, Goping G, and Mueller GP. Proteomic analysis of rat atrial secretory granules: a platform for testable hypotheses. Biochim Biophys Acta 2004; 1699: 263 - 275