【作者】 张秀花；

【导师】 苏定冯；

【作者基本信息】 第二军医大学 ， 药理学， 2009， 博士

【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。更多还原

【Abstract】 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.更多还原