参与脑缺血损伤和保护机制的蛋白靶点筛查
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摘要
脑血管疾病是指供应脑部血液的血管的疾病所致的一种神经系统疾病，以其高发病率、高死亡率、高致残率、高复发率成为严重威胁人类生命与健康的主要疾病之一，主要包括缺血性和出血性脑卒中，其中缺血性脑卒中最常见，约占70％-80％。目前，脑卒中的有效治疗药物十分单一，并且因为苛刻的治疗时间窗和细胞毒副作用而使其应用受到限制。脑卒中的临床治疗急切需要新的理论基础和分子靶点。
脑卒中在发生后的几小时到几天内就会有大量复杂的病理生理变化。在缺血中心区，血流缺失、ATP和能量储存低水平、离子紊乱和代谢障碍都很严重，细胞在几分钟内就死亡了。但是在中心区的周围——半影带——因为侧枝循环而仍然存在少量血流，损伤也因而轻微些，通常表现为凋亡样的迟发性神经元死亡。因此，针对半影带细胞相对慢速的主动死亡机制在临床上发展治疗手段是合理可行的选择。为了找到新的参与细胞损伤(CA1区损伤后上调的和CA3区损伤后下调的蛋白)和内源性保护(CA3区损伤后上调的和CAl区损伤后下调的蛋白)的分子机制，本研究在短暂性全脑缺血神经元迟发性死亡模型上，选择缺血损伤敏感性海马CA1区及其相邻的缺血损伤非敏感性CA3区进行了蛋白表达水平观察。
脑卒中过程中细胞的死亡至少有三个基本的机制：兴奋性毒性和离子失平衡，氧化／亚硝化应激，凋亡样的细胞死亡。这些机制之间是相互交叉，而非孤立的；作用对象也是广泛的，包括了神经细胞、胶质细胞和血管元件；在亚细胞水平上，它们涉及线粒体、核、细胞膜、内质网和溶酶体等多个细胞器已知一些蛋白分子(包括离子通道、线粒体呼吸链成分、细胞内信号转导分子)的表达变化参与甚至决定了这些机制的进行。在以上这些主要已知的细胞死亡途径中，线粒体是已知最重要的细胞器之一。线粒体包含了一系列促凋亡因子，包括细胞色素c、继发性线粒体源性caspase激活因子(secondary mitochondrial activator of caspase，Smac／Diablo)、核酸内切酶、AIF等，线粒体上转运孔道的形成，氧化磷酸化相关成分的变化都是线粒体成为关键细胞器的原因。为了找到新的参与细胞损伤或者保护机制的线粒体组分，本研究在短暂性全脑缺血神经元迟发性死亡模型上，提取纯化线粒体后选择观察了缺血前后线粒体蛋白表达量的变化。
在现有的蛋白质组学技术平台中，双向电泳(two dimensional electrophoresis，2DE)技术已经是一种成熟的蛋白分离技术，因其能够同时分离和显示数以千计的蛋白这一独特优势，以及相关仪器和试剂成本相对低廉，成为大多数研究者的首选。2004年新发明的“Silver Blue”染色方法是对1988年Neuhoff方法的改进，这种胶体考马斯亮蓝G-250染色法已经能达到ng级敏感度，改变了以往mg级上样量也只能得到不足一千个点的状况。应用这种方法，我们在1mg总上样量时常能得到两千余蛋白斑点。并且其很好的可重复性是银染很难达到的。研究采用的质谱方法是基质辅助激光解吸电离-飞行时间质谱(matrix assisted laser desorption／ionization time-of-flight mass spectrometry，MALDI-TOF-MS)和时间串联质谱(MALDI-TOF／TOF-MS)，后者可以将酶解肽段进一步解离成次第减少一个氨基酸的肽段，从而实现从头测序(de novo sequencing)，得到多个选检肽段的完整氨基酸序列信息，进而推断蛋白质序列信息，提高了待测蛋白的检出成功率和准确率。
以往有多项研究观察脑缺血损伤相关的基因和蛋白表达变化，所使用的技术方法包括寡核苷酸芯片、表达序列标签cDNA芯片、高通量的免疫印迹法、双向电泳联用可定量的同位素标记亲和标签、表面增强激光解离／离子化-时间飞跃质谱等，研究材料多为全脑、全海马组织、培养皮层细胞等，也有研究在间歇性缺氧的大鼠模型上，对缺氧损伤敏感性截然不同的CA1和CA3区采用2DE-MS筛选到一些骨架蛋白、应激相关蛋白、凋亡相关蛋白变化，但是尚未见到使用蛋白质组学方法对CA1和CA3区、海马组织线粒体在脑缺血后变化规律进行研究的报道。本研究的技术路线主要依靠双向电泳和质谱技术筛选可能的候选靶分子，并重点选择进行了免疫学的验证。
假手术组和四血管夹闭模型导致大脑缺血再灌注损伤的大鼠被经左心室灌注固定后，制作海马组织冰冻切片并进行神经元特异的尼氏染色，随后对存活神经元(胞体饱满、染色均匀者)进行计数，比较损伤后24小时和7天是否比假手术组有神经元数量变化。发现CA1区神经元密度在损伤后24小时(181.72±1.50，n=6)和7天(28.74±0.93，n=7)都较假手术组(203.70±2.72，n=4)降低，结果有显著性差异(单向方差分析，F=3950，P=0.000)。而CA3区神经元密度则无明显变化。由于7天后CA1区大量神经元已经死亡，研究此时神经元内蛋白表达水平对阐明死亡发生前信号机制不再有帮助。所以选择24小时作为研究的时间点，此时大部分神经元尚存活，但是损伤的作用已经开始显现。从假手术组大鼠分离得到海马组织CA1和CA3分区，制备成蛋白样品，经双向电泳，凝胶固定、染色、脱色，图像扫描后得到蛋白分离良好、高度可重复的组织蛋白双向电泳图。应用专业图像分析软件对假手术组CA1和CA3差异表达的蛋白进行分析筛选，最后挑选出9个蛋白进行了MALDI-TOF／TOF-MS鉴定，成功鉴定出8个。对脑缺血24小时后CA1和CA3分别发生差异表达的蛋白进行分析筛选，最后挑选出23个蛋白进行了MALDI-TOF／TOF-MS鉴定，成功鉴定出21个。从新鲜的假手术组和脑缺血损伤的大鼠海马组织中分离提纯获得线粒体后，使用电子显微镜检视分离出的海马组织线粒体。大鼠海马组织线粒体被固定、切片、染色后进行电子显微镜观察，可见大部分提纯物(约占70～80％)为线粒体，并且线粒体结构完整，膜和嵴保存完好。从假手术组和脑缺血24小时后的大鼠分离得到海马组织线粒体，制备成蛋白样品，经双向电泳，凝胶固定、染色、脱色，图像扫描后得到蛋白分离良好、高度可重复的组织蛋白双向电泳图。应用专业图像分析软件PDQest 7．0对脑缺血24小时后海马组织线粒体发生差异表达的蛋白进行分析筛选，最后挑选出24个蛋白进行了MALDI-TOF-MS鉴定，成功鉴定出13个，其中抑制素在脑缺血24小时后发生上调。为了验证海马组织线粒体中抑制素在脑缺血后发生上调的变化，并细致探究其变化的时相规律，应用免疫印迹技术探测了假手术后24小时和脑缺血损伤后1、6、12、24小时的抑制素表达水平。结果发现与假手术组相比，海马组织线粒体中抑制素在脑缺血损伤后1小时尚未发生变化，6小时后累积量升高，差异有显著性意义(单向方差分析，F=9.642，p=0.002)。
本研究主要采用以双向电泳联合飞行时间(串联)质谱为技术平台的蛋白质组学方法，结合脑缺血的特异脑区损伤易感性差异现象和重要损伤机制靶细胞器，筛选出了一些和脑缺血损伤、保护相关的蛋白分子靶点。在假手术组大鼠海马组织中，我们发现CA1和CA3区蛋白质组存在差异表达。成功鉴定的8个蛋白分别是胶质细胞纤维酸性蛋白d(glial fibrillary acidic protein delta，GFAP d)，磷酸甘油醛异构酶(triose phosphate isomerase 1，Tpil)，海兔ras相关同系物A2(aplysia ras-related homolog A2)，大鼠神经生长抑制因子反应介导蛋白3(rat Collapsin response mediator proteins-3，rCRMP-3)，高迁移率族蛋白B1(High mobility group box 1，HMGBl)，血小板活化因子乙酰水解酶异构体1b的a2片段(platelet-activating factor acetylhydrolase isoform 1b，alpha2)，胆绿素还原酶样蛋白(similar to biliverdin reductase)，谷氨酸盐合成酶1(Glutamine synthetase 1)。其中谷氨酸盐合成酶1在CA3区高表达，余者皆高表达于CA1区。大鼠神经生长抑制因子反应介导蛋白3有可能成为缺血后神经再生的标志之一。磷酸甘油醛异构酶在CA1区的高表达可能是其易感性高的分子基础之一。谷氨酸盐合成酶1对于及时消除可能过剩的公认神经兴奋性毒性物质谷氨酸很有利，理论上能提高CA3的抗损伤能力。脑缺血再灌注损伤24小时后，我们发现大鼠海马组织CA1蛋白质组存在差异表达。成功鉴定的蛋白中损伤后下调的蛋白有热休克蛋白60家族成员(未命名)，肌酸激酶B(creatine kinase B，CKB)，LIM and SH3 protein1(LASPl)，ATP合酶，brain abundant，membrane attached signal protein 1，胆绿素还原酶样蛋白；损伤后上调的蛋白有磷酸甘油酸盐变位酶1(phosphoglycerate mutase 1，PGAM1)，应激诱生磷酸化蛋白1(或称热休克蛋白68／Hsc70和热休克蛋白90组织蛋白)，alpha B-晶状体球蛋白，DJ-1。肌酸激酶B在梗塞灶(如CA1)的作用和变化并未见报道，推测是梗塞灶的能量障碍原因之一。ATP合酶是合成重要能量物质ATP的核心酶，它的下调可能是梗塞灶能量障碍的原因之一。PGAM1的上调提示梗塞灶中糖酵解水平上升或者因为能量障碍导致酶的反应性上调，并且可能是脑卒中损伤的治疗靶点。alpha B-晶状体球蛋白在心肌缺血中的抗凋亡机制是否在脑缺血模型中同样存在、程度如何都有待进一步研究。血清DJ-1可以作为脑卒中的早期发病标志物，但在CA1梗塞灶的作用未定。脑缺血再灌注损伤24小时后，我们发现大鼠海马组织CA3蛋白质组存在差异表达。成功鉴定的蛋白中损伤后下调的蛋白有二磷酸核苷激酶；损伤后上调的蛋白有，电压依赖阴离子通道(voltage-dependent anion channel，VDAC)样蛋白，脑肌酸激酶，鸟嘌呤脱氨酶，脑血影蛋白a链(brain Spectrin alpha chain，SPNA2)，alphasynuclein，ATP合酶beta~基，大鼠肝脏FIATP酶B链。VDAC可能是渗透性转换孔道复合体(permeability transition pore complex，PTPC)的组分，参与释放一些促凋亡的线粒体成分来影响脑卒中进程。以往未见二磷酸核菅激酶等在CA3区发生表达变化和功能的报道。
脑缺血再灌注损伤24小时后，我们发现大鼠海马组织线粒体蛋白质组存在差异表达。成功鉴定的蛋白中损伤后上调的有热休克蛋白70 1A／1B，Alpha-微管蛋白3，抑制素，突触素-2，Heat shock cognate 71 kDa protein，鸟嘌呤结合蛋白beta亚基1(Transducin beta亚基1)，下调的有海马胆碱能神经激肽(Hippocampal cholinergic neurostimulating peptide，HCNP)，丙酮酸脱氢酶1 alpha亚基(Pyruvate dehydrogenase E1 component alpha subunit，PDHE1-A typeⅠ)，LIMand SH3 domain protein 1(LASP-1)，ATP合酶beta亚基，Alpha-internexin，谷胱甘肽S-转移酶p，胶质细胞成熟因子beta(Glia maturation factor beta，GMF-beta)。丙酮酸脱氢酶是三羧酸循环的关键酶之一，ATP合酶是合成重要能量物质ATP的酶，它们的下调势必影响到梗塞灶的能量供给。谷胱甘肽S-转移酶p作为卒中相关基因可能通过抑制JNK和STAT通路来保护脑缺血。脑缺血后上调的线粒体蛋白中抑制素(prohibitin)在既往研究中主要影响生殖系统细胞增殖和凋亡，是有丝分裂Rb-E2F关键通路中的重要组分。近来有研究证明了许多疾病中成熟神经元死亡前会有异常的细胞周期启动，最近还有研究显示抑制素参与了氧化应激损伤，更有趣的是它的凋亡相关功能和它在线粒体与核之间的穿梭有联系，提示这一蛋白的功能表现可能提供了线粒体和核之间对话的新例证并参与缺血缺氧性脑病(比如脑卒中)的机制。我们还需要进一步干预prohibitin的整体表达量或者线粒体内累积量，观察是否影响神经细胞的存活，及其相关信号分子的表达和功能水平。
Cerebral vascular diseases, nervous system diseases due to disabilities of cerebral vessels, are among those most popular diseases with high incidence, mortality rate, disabilities rate and recurrence rate. Ischemic stroke is the most popular type of cerebral vascular diseases, accounting for 70%-80% of them. Ischemic stroke is characterized by complex spatial and temporal events evolving over hours or even days. In the core ischemic territory, blood flow deficits, low ATP levels and energy stores, ionic disruption and metabolic failure are severe, and cell death progresses in minutes. However, the peripheral zones within the flow compromised territory—the ischemic penumbra—suffer milder insults due to residual perfusion from collateral blood vessels. Since relatively slow progress of active cell death in penumbra, it is reasonable to develop clinic therapy aiming at this area. The present study compared the proteome of hippocampal CA1 and its nature internal control CA3 neighboring subregions with distinct susceptibility to transient global ischemia, assuming that the proteomic difference indicates injury, protection and regeneration molecular mechanisms. There are at least three fundamental mechanisms leading to cell death during ischemic brain injury: excitotoxicity and ionic imbalance, oxidative/nitrosative stress, and apoptotic-like cell death. These mechanisms demonstrate overlapping and redundant features. They mediate injury within neurons, glia and vascular elements, and at the subcellular level, they impact the function of mitochondria, nuclei, cell membranes, endoplasmic reticula and lysosomes. Some known molecules are key factors in these pathways, such as ion channels, mitochondrial apoptotic components and nucleic transcription factors. Mitochondria and nuclei are the most important organelles involved in the above mechanisms. Mitochondria contain a number of proapoptotic molecules, including cytochrome c, secondary mitochondrial activator of caspase(Smac/Diablo), endonulease, apoptosis inducing-factor(AIF), and so on. Formation of permeable transition pore on mitochondrial membranes as an intial step of apoptosis makes this organelle the core of cell fate. Nuclei are not only the targets of injury, but the origin of protein synthesis involved in death and survival pathways as well. Up to now, mitochondria are considered mainly as the upstream of nuclei. Our present study profiled the mitochondrial proteomic variation after stroke to find new target molecules involved in cell injury and protection mechanisms. Two dimensional electrophoresis (2DE), the favourite of most proteomics researchers, has unique priority of separating and visualizing thousands of protein spots simultaneously by cheap apparatus and reagents. The "Silver Blue" staining method developed after Neuhoff's was announced to have achieved the sensitivity of nanogram, assuring more than a thousands of spots routinely after 1 mg of loading. We have obtained about 2000 spots of lmg tissue protein with much better reproducibility than silver staining. The biological mass spectrometry we've chosen were matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/TOF MS. The TOF/TOF MS can deduce protein identity by de novo sequencing of the further dissociated ions after MALDI, which has much more hits than MALDI-TOF-MS. There have been a number of genomic and proteomic investigation on brain ischemia (whole brain, hippocampus or cultured cortex), employing oligonucleotide array, expressed sequencing tags (EST) cDNA array, high-throughput immunoblot, 2-DE tandem isotope coded affinity tags(ICAT), surface enhanced laser dissociation/ionization time-of-flight mass spectrometry (SELDI-TOF). 2DE-MS has been implemented to identify proteins differentially expressed in the CA1 and CA3 regions of the rat hippocampus and to assess changes in protein expression following a 6-h exposure to intermittent hypoxia. Proteomic investigation on CA1 and CA3 regions or mitochondria enduring ischemic stroke has not been tried as much as we know. The present study was mainly based upon 2-DE tandem mass spectrometry to pan out hippocampal subregions specific or mitochondrial targets involved in stroke, which was verified by immunological technique later.
Rats after sham control operation and four-vessel occlusion (4-VO) of ischemia-reperfusion injury were transcardiacally perfused and fixed. Neurons were visualized by nissel staining on frozen hippocampus slices to examine the survival extent after operation at time points of 24 hours and 7 days. After injury, CA1 neurons began to die after 24hr (181.72±1.50), and collapsed broadly after 7 days (28.74±0.93), much less than the sham control (203.70±2.72) with statistical significance. Nevertheless CA3 neurons kept intact all along (table 1). Research on CA1 proteome 7 days after stroke when broad cell death had occurred was useless to reveal the molecular mechanisms before death. We chose 24hour after stroke when injury effect on neuron had begun before their death as the time point versus sham control of observation. Brain coronal sections were acutely obtained from sham control animals, followed by dissociation of hippocampal subregions CA1 and CA3. Tissue was prepared before two dimensional electrophoresis, gel staining and image scanning to obtain gel images with good separation and reproducibility. Differentially displayed proteins between sham control CA1 and CA3 were revealed by analysis of professional software. 8 of those 9 picked protein spots were successfully sequenced by MALDI-TOF/TOF-MS. Proteins differentially expressed between the sham control and 24hr stroke groups were also uncovered in the same way, and 21 of those 23 picked protein spots were successfully sequenced. Hippocampal mitochondria were separated from rat of sham control group and 24h after stroke group, and then examined by electron microscopy. The mitochondria were fixed, sliced, stained and observed, A large proportion of (70-80%) extracts were mitochondria with intact membrane and cristae. Mitochondrial proteins differentially expressed between the sham control and 24hr stroke groups were also uncovered in the same way, except for MALDI-TOF-MS instead of MALDI-TOF/TOF-MS, and 13 of those 24 picked protein spots were successfully sequenced. Among the sequenced mitochondrial proteins, prohibitin was up regulated after stroke and validated by immunoblot to scale its dynamics (1, 6, 12, 24hour after stroke). Immunoblot results shew that accumulation of mitochondrial prohibitin didn't change lhour after stroke, but was up regulated 6hours after stroke (one-ANOVA, p p＜0.05, n=3).
The present study mined according to uniquely area specific susceptibility and key role of mitochondria in cell injury for molecular targets involved in stroke, mainly based upon the proteomic technologies of 2-DE tandem MS-TOF(/TOF)-MS. We found difference of proteome between sham control CA1 and CA3. The 8 successfully identified proteins were GFAP d (glial fibrillary acidic protein delta), Tpil (triose phosphate isomerase 1), aplysia ras-related homolog A2, rCRMP-3 (rat Collapsin response mediator proteins-3), HMGB1 (High mobility group box 1), platelet-activating factor acetylhydrolase isoform lb alpha2, protein similar to biliverdin reductase, glutamine synthetase 1。Glutamine synthetase 1 was more highly expressed in CA3, the others were more in CA1. rCRMP-3 may be a marker of postischemic neurogenesis. Tpil may contribute to higher susceptibility of CA1 for its higher expression in this area. Glutamine synthetase 1 can reasonably eliminate excess neurotoxic glutamate in CA3 to protect this area from stroke injury. We found differentially expressed proteins in CA1 after 24 hours of brain ischemia-reperfusion injury. The successfully identified proteins included down regulated unnamed protein product (HSP60 family), CKB (creatine kinase B), LASPI(LIM and SH3 protein 1), ATP synthase, brain abundant, membrane attached signal protein 1, protein similar to biliverdin reductase, and up regulated PGAM1 (phosphoglycerate mutase 1), stress-induced-phosphoprotein 1 (Hsp70/Hsp90-organizing protein), alpha B-crystallin, and DJ-1 protein. Creatine kinase B was not reported to be involved in ischemic area. We speculate that its role as a metabolic enzyme contribute to energy deficiency there. ATP synthase is the core enzyme to synthesize ATP, so its down regulation reasonably causes energy deficiency in ischemic area. Upregulation of PGAM1 indicated the increased glycolysis or energy deficiency in ischemic area; itself can be a potential therapeutic target. Whether the antiapoptotic role of alpha B-crystallin in myocarcial ischemia is also present in brain requires further investigation. Serum DJ-1 has already been considered as early marker of brain ischemia stroke, although its detailed local function in ischemic area remain unclear. We found differentially expressed proteins in CA3 after 24 hours of brain ischemia-reperfusion injury. The successfully identified proteins included down regulated nucleoside diphosphate kinase and upregulated voltage-dependent anion channel-like protein, brain creatine kinase, guanine deaminase, SPNA2 (brain Spectrin alpha chain), alpha synuclein, ATP synthase beta subunit, Rat Liver F1-Atpase Chain B. VDAC is included in the PTPC (permeability transition pore complex) which is proved to allow some proapoptotic components to release, so VDAC may have some effect on progress of brain ischemia. There was no report about role of nucleoside diphosphate kinase and so on in brain ischemia as much as we know. Some mitochondrial proteins in hippocampus were found to be differentially expressed after 24 hours of brain ischemia-reperfusion injury. The successfully identified proteins included down regulated HCNP (Hippocampal cholinergic neurostimulating peptide), PDHE1-A type I (Pyruvate dehydrogenase E1 component alpha subunit), LASP-1 (LIM and SH3 domain protein 1), ATP synthase beta subunit, Alpha-internexin, GSTp (Glutathione S-transferase pi), GMF-beta (Glia maturation factor beta), and upregulated Heat shock 70 kDa protein 1A/1B, Alpha-tubulin 3, Prohibitin, Synapsin-2, Heat shock cognate 71 kDa protein, and Guanine nucleotide-binding protein subunit beta 1. PDH (Pyruvate dehydrogenase) is key enzyme in TCA cycle (tricarboxylic acid cycle). Downregulation of PDH and ATP synthase would centainly interrupt the energy supply. GSTp previously implicated as stroke related gene's product may play protective role through inhibition of JNK and STAT pathways. Preceding research on prohibitin was primarily settled in productive organs and their derived carcinomas. Prohibitin was proved to be a key component of Rb-E2F pathway which plays the central role in mitosis. Recent studies have proved importance of aberrant cell cycle reentry before various nervous system diseases. Prohibitin has been shown to be involved in oxidative stress injury, which may due to its shuttle between mitochondria and nuclei. Our research can provided a new proof of crosstalk between those two organelles by prohibitin and its role in ischemic hypoxic brain diseases such as ischemic stroke. To verify the above hypothesis, we need to interfere the cellular expression and mitochondrial accumulation of prohibitin, and observe the survival of neurons and expression and activity of prohibitin related signal molecules
脑卒中在发生后的几小时到几天内就会有大量复杂的病理生理变化。在缺血中心区，血流缺失、ATP和能量储存低水平、离子紊乱和代谢障碍都很严重，细胞在几分钟内就死亡了。但是在中心区的周围——半影带——因为侧枝循环而仍然存在少量血流，损伤也因而轻微些，通常表现为凋亡样的迟发性神经元死亡。因此，针对半影带细胞相对慢速的主动死亡机制在临床上发展治疗手段是合理可行的选择。为了找到新的参与细胞损伤(CA1区损伤后上调的和CA3区损伤后下调的蛋白)和内源性保护(CA3区损伤后上调的和CAl区损伤后下调的蛋白)的分子机制，本研究在短暂性全脑缺血神经元迟发性死亡模型上，选择缺血损伤敏感性海马CA1区及其相邻的缺血损伤非敏感性CA3区进行了蛋白表达水平观察。
脑卒中过程中细胞的死亡至少有三个基本的机制：兴奋性毒性和离子失平衡，氧化／亚硝化应激，凋亡样的细胞死亡。这些机制之间是相互交叉，而非孤立的；作用对象也是广泛的，包括了神经细胞、胶质细胞和血管元件；在亚细胞水平上，它们涉及线粒体、核、细胞膜、内质网和溶酶体等多个细胞器已知一些蛋白分子(包括离子通道、线粒体呼吸链成分、细胞内信号转导分子)的表达变化参与甚至决定了这些机制的进行。在以上这些主要已知的细胞死亡途径中，线粒体是已知最重要的细胞器之一。线粒体包含了一系列促凋亡因子，包括细胞色素c、继发性线粒体源性caspase激活因子(secondary mitochondrial activator of caspase，Smac／Diablo)、核酸内切酶、AIF等，线粒体上转运孔道的形成，氧化磷酸化相关成分的变化都是线粒体成为关键细胞器的原因。为了找到新的参与细胞损伤或者保护机制的线粒体组分，本研究在短暂性全脑缺血神经元迟发性死亡模型上，提取纯化线粒体后选择观察了缺血前后线粒体蛋白表达量的变化。
在现有的蛋白质组学技术平台中，双向电泳(two dimensional electrophoresis，2DE)技术已经是一种成熟的蛋白分离技术，因其能够同时分离和显示数以千计的蛋白这一独特优势，以及相关仪器和试剂成本相对低廉，成为大多数研究者的首选。2004年新发明的“Silver Blue”染色方法是对1988年Neuhoff方法的改进，这种胶体考马斯亮蓝G-250染色法已经能达到ng级敏感度，改变了以往mg级上样量也只能得到不足一千个点的状况。应用这种方法，我们在1mg总上样量时常能得到两千余蛋白斑点。并且其很好的可重复性是银染很难达到的。研究采用的质谱方法是基质辅助激光解吸电离-飞行时间质谱(matrix assisted laser desorption／ionization time-of-flight mass spectrometry，MALDI-TOF-MS)和时间串联质谱(MALDI-TOF／TOF-MS)，后者可以将酶解肽段进一步解离成次第减少一个氨基酸的肽段，从而实现从头测序(de novo sequencing)，得到多个选检肽段的完整氨基酸序列信息，进而推断蛋白质序列信息，提高了待测蛋白的检出成功率和准确率。
以往有多项研究观察脑缺血损伤相关的基因和蛋白表达变化，所使用的技术方法包括寡核苷酸芯片、表达序列标签cDNA芯片、高通量的免疫印迹法、双向电泳联用可定量的同位素标记亲和标签、表面增强激光解离／离子化-时间飞跃质谱等，研究材料多为全脑、全海马组织、培养皮层细胞等，也有研究在间歇性缺氧的大鼠模型上，对缺氧损伤敏感性截然不同的CA1和CA3区采用2DE-MS筛选到一些骨架蛋白、应激相关蛋白、凋亡相关蛋白变化，但是尚未见到使用蛋白质组学方法对CA1和CA3区、海马组织线粒体在脑缺血后变化规律进行研究的报道。本研究的技术路线主要依靠双向电泳和质谱技术筛选可能的候选靶分子，并重点选择进行了免疫学的验证。
假手术组和四血管夹闭模型导致大脑缺血再灌注损伤的大鼠被经左心室灌注固定后，制作海马组织冰冻切片并进行神经元特异的尼氏染色，随后对存活神经元(胞体饱满、染色均匀者)进行计数，比较损伤后24小时和7天是否比假手术组有神经元数量变化。发现CA1区神经元密度在损伤后24小时(181.72±1.50，n=6)和7天(28.74±0.93，n=7)都较假手术组(203.70±2.72，n=4)降低，结果有显著性差异(单向方差分析，F=3950，P=0.000)。而CA3区神经元密度则无明显变化。由于7天后CA1区大量神经元已经死亡，研究此时神经元内蛋白表达水平对阐明死亡发生前信号机制不再有帮助。所以选择24小时作为研究的时间点，此时大部分神经元尚存活，但是损伤的作用已经开始显现。从假手术组大鼠分离得到海马组织CA1和CA3分区，制备成蛋白样品，经双向电泳，凝胶固定、染色、脱色，图像扫描后得到蛋白分离良好、高度可重复的组织蛋白双向电泳图。应用专业图像分析软件对假手术组CA1和CA3差异表达的蛋白进行分析筛选，最后挑选出9个蛋白进行了MALDI-TOF／TOF-MS鉴定，成功鉴定出8个。对脑缺血24小时后CA1和CA3分别发生差异表达的蛋白进行分析筛选，最后挑选出23个蛋白进行了MALDI-TOF／TOF-MS鉴定，成功鉴定出21个。从新鲜的假手术组和脑缺血损伤的大鼠海马组织中分离提纯获得线粒体后，使用电子显微镜检视分离出的海马组织线粒体。大鼠海马组织线粒体被固定、切片、染色后进行电子显微镜观察，可见大部分提纯物(约占70～80％)为线粒体，并且线粒体结构完整，膜和嵴保存完好。从假手术组和脑缺血24小时后的大鼠分离得到海马组织线粒体，制备成蛋白样品，经双向电泳，凝胶固定、染色、脱色，图像扫描后得到蛋白分离良好、高度可重复的组织蛋白双向电泳图。应用专业图像分析软件PDQest 7．0对脑缺血24小时后海马组织线粒体发生差异表达的蛋白进行分析筛选，最后挑选出24个蛋白进行了MALDI-TOF-MS鉴定，成功鉴定出13个，其中抑制素在脑缺血24小时后发生上调。为了验证海马组织线粒体中抑制素在脑缺血后发生上调的变化，并细致探究其变化的时相规律，应用免疫印迹技术探测了假手术后24小时和脑缺血损伤后1、6、12、24小时的抑制素表达水平。结果发现与假手术组相比，海马组织线粒体中抑制素在脑缺血损伤后1小时尚未发生变化，6小时后累积量升高，差异有显著性意义(单向方差分析，F=9.642，p=0.002)。
本研究主要采用以双向电泳联合飞行时间(串联)质谱为技术平台的蛋白质组学方法，结合脑缺血的特异脑区损伤易感性差异现象和重要损伤机制靶细胞器，筛选出了一些和脑缺血损伤、保护相关的蛋白分子靶点。在假手术组大鼠海马组织中，我们发现CA1和CA3区蛋白质组存在差异表达。成功鉴定的8个蛋白分别是胶质细胞纤维酸性蛋白d(glial fibrillary acidic protein delta，GFAP d)，磷酸甘油醛异构酶(triose phosphate isomerase 1，Tpil)，海兔ras相关同系物A2(aplysia ras-related homolog A2)，大鼠神经生长抑制因子反应介导蛋白3(rat Collapsin response mediator proteins-3，rCRMP-3)，高迁移率族蛋白B1(High mobility group box 1，HMGBl)，血小板活化因子乙酰水解酶异构体1b的a2片段(platelet-activating factor acetylhydrolase isoform 1b，alpha2)，胆绿素还原酶样蛋白(similar to biliverdin reductase)，谷氨酸盐合成酶1(Glutamine synthetase 1)。其中谷氨酸盐合成酶1在CA3区高表达，余者皆高表达于CA1区。大鼠神经生长抑制因子反应介导蛋白3有可能成为缺血后神经再生的标志之一。磷酸甘油醛异构酶在CA1区的高表达可能是其易感性高的分子基础之一。谷氨酸盐合成酶1对于及时消除可能过剩的公认神经兴奋性毒性物质谷氨酸很有利，理论上能提高CA3的抗损伤能力。脑缺血再灌注损伤24小时后，我们发现大鼠海马组织CA1蛋白质组存在差异表达。成功鉴定的蛋白中损伤后下调的蛋白有热休克蛋白60家族成员(未命名)，肌酸激酶B(creatine kinase B，CKB)，LIM and SH3 protein1(LASPl)，ATP合酶，brain abundant，membrane attached signal protein 1，胆绿素还原酶样蛋白；损伤后上调的蛋白有磷酸甘油酸盐变位酶1(phosphoglycerate mutase 1，PGAM1)，应激诱生磷酸化蛋白1(或称热休克蛋白68／Hsc70和热休克蛋白90组织蛋白)，alpha B-晶状体球蛋白，DJ-1。肌酸激酶B在梗塞灶(如CA1)的作用和变化并未见报道，推测是梗塞灶的能量障碍原因之一。ATP合酶是合成重要能量物质ATP的核心酶，它的下调可能是梗塞灶能量障碍的原因之一。PGAM1的上调提示梗塞灶中糖酵解水平上升或者因为能量障碍导致酶的反应性上调，并且可能是脑卒中损伤的治疗靶点。alpha B-晶状体球蛋白在心肌缺血中的抗凋亡机制是否在脑缺血模型中同样存在、程度如何都有待进一步研究。血清DJ-1可以作为脑卒中的早期发病标志物，但在CA1梗塞灶的作用未定。脑缺血再灌注损伤24小时后，我们发现大鼠海马组织CA3蛋白质组存在差异表达。成功鉴定的蛋白中损伤后下调的蛋白有二磷酸核苷激酶；损伤后上调的蛋白有，电压依赖阴离子通道(voltage-dependent anion channel，VDAC)样蛋白，脑肌酸激酶，鸟嘌呤脱氨酶，脑血影蛋白a链(brain Spectrin alpha chain，SPNA2)，alphasynuclein，ATP合酶beta~基，大鼠肝脏FIATP酶B链。VDAC可能是渗透性转换孔道复合体(permeability transition pore complex，PTPC)的组分，参与释放一些促凋亡的线粒体成分来影响脑卒中进程。以往未见二磷酸核菅激酶等在CA3区发生表达变化和功能的报道。
脑缺血再灌注损伤24小时后，我们发现大鼠海马组织线粒体蛋白质组存在差异表达。成功鉴定的蛋白中损伤后上调的有热休克蛋白70 1A／1B，Alpha-微管蛋白3，抑制素，突触素-2，Heat shock cognate 71 kDa protein，鸟嘌呤结合蛋白beta亚基1(Transducin beta亚基1)，下调的有海马胆碱能神经激肽(Hippocampal cholinergic neurostimulating peptide，HCNP)，丙酮酸脱氢酶1 alpha亚基(Pyruvate dehydrogenase E1 component alpha subunit，PDHE1-A typeⅠ)，LIMand SH3 domain protein 1(LASP-1)，ATP合酶beta亚基，Alpha-internexin，谷胱甘肽S-转移酶p，胶质细胞成熟因子beta(Glia maturation factor beta，GMF-beta)。丙酮酸脱氢酶是三羧酸循环的关键酶之一，ATP合酶是合成重要能量物质ATP的酶，它们的下调势必影响到梗塞灶的能量供给。谷胱甘肽S-转移酶p作为卒中相关基因可能通过抑制JNK和STAT通路来保护脑缺血。脑缺血后上调的线粒体蛋白中抑制素(prohibitin)在既往研究中主要影响生殖系统细胞增殖和凋亡，是有丝分裂Rb-E2F关键通路中的重要组分。近来有研究证明了许多疾病中成熟神经元死亡前会有异常的细胞周期启动，最近还有研究显示抑制素参与了氧化应激损伤，更有趣的是它的凋亡相关功能和它在线粒体与核之间的穿梭有联系，提示这一蛋白的功能表现可能提供了线粒体和核之间对话的新例证并参与缺血缺氧性脑病(比如脑卒中)的机制。我们还需要进一步干预prohibitin的整体表达量或者线粒体内累积量，观察是否影响神经细胞的存活，及其相关信号分子的表达和功能水平。
Cerebral vascular diseases, nervous system diseases due to disabilities of cerebral vessels, are among those most popular diseases with high incidence, mortality rate, disabilities rate and recurrence rate. Ischemic stroke is the most popular type of cerebral vascular diseases, accounting for 70%-80% of them. Ischemic stroke is characterized by complex spatial and temporal events evolving over hours or even days. In the core ischemic territory, blood flow deficits, low ATP levels and energy stores, ionic disruption and metabolic failure are severe, and cell death progresses in minutes. However, the peripheral zones within the flow compromised territory—the ischemic penumbra—suffer milder insults due to residual perfusion from collateral blood vessels. Since relatively slow progress of active cell death in penumbra, it is reasonable to develop clinic therapy aiming at this area. The present study compared the proteome of hippocampal CA1 and its nature internal control CA3 neighboring subregions with distinct susceptibility to transient global ischemia, assuming that the proteomic difference indicates injury, protection and regeneration molecular mechanisms. There are at least three fundamental mechanisms leading to cell death during ischemic brain injury: excitotoxicity and ionic imbalance, oxidative/nitrosative stress, and apoptotic-like cell death. These mechanisms demonstrate overlapping and redundant features. They mediate injury within neurons, glia and vascular elements, and at the subcellular level, they impact the function of mitochondria, nuclei, cell membranes, endoplasmic reticula and lysosomes. Some known molecules are key factors in these pathways, such as ion channels, mitochondrial apoptotic components and nucleic transcription factors. Mitochondria and nuclei are the most important organelles involved in the above mechanisms. Mitochondria contain a number of proapoptotic molecules, including cytochrome c, secondary mitochondrial activator of caspase(Smac/Diablo), endonulease, apoptosis inducing-factor(AIF), and so on. Formation of permeable transition pore on mitochondrial membranes as an intial step of apoptosis makes this organelle the core of cell fate. Nuclei are not only the targets of injury, but the origin of protein synthesis involved in death and survival pathways as well. Up to now, mitochondria are considered mainly as the upstream of nuclei. Our present study profiled the mitochondrial proteomic variation after stroke to find new target molecules involved in cell injury and protection mechanisms. Two dimensional electrophoresis (2DE), the favourite of most proteomics researchers, has unique priority of separating and visualizing thousands of protein spots simultaneously by cheap apparatus and reagents. The "Silver Blue" staining method developed after Neuhoff's was announced to have achieved the sensitivity of nanogram, assuring more than a thousands of spots routinely after 1 mg of loading. We have obtained about 2000 spots of lmg tissue protein with much better reproducibility than silver staining. The biological mass spectrometry we've chosen were matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/TOF MS. The TOF/TOF MS can deduce protein identity by de novo sequencing of the further dissociated ions after MALDI, which has much more hits than MALDI-TOF-MS. There have been a number of genomic and proteomic investigation on brain ischemia (whole brain, hippocampus or cultured cortex), employing oligonucleotide array, expressed sequencing tags (EST) cDNA array, high-throughput immunoblot, 2-DE tandem isotope coded affinity tags(ICAT), surface enhanced laser dissociation/ionization time-of-flight mass spectrometry (SELDI-TOF). 2DE-MS has been implemented to identify proteins differentially expressed in the CA1 and CA3 regions of the rat hippocampus and to assess changes in protein expression following a 6-h exposure to intermittent hypoxia. Proteomic investigation on CA1 and CA3 regions or mitochondria enduring ischemic stroke has not been tried as much as we know. The present study was mainly based upon 2-DE tandem mass spectrometry to pan out hippocampal subregions specific or mitochondrial targets involved in stroke, which was verified by immunological technique later.
Rats after sham control operation and four-vessel occlusion (4-VO) of ischemia-reperfusion injury were transcardiacally perfused and fixed. Neurons were visualized by nissel staining on frozen hippocampus slices to examine the survival extent after operation at time points of 24 hours and 7 days. After injury, CA1 neurons began to die after 24hr (181.72±1.50), and collapsed broadly after 7 days (28.74±0.93), much less than the sham control (203.70±2.72) with statistical significance. Nevertheless CA3 neurons kept intact all along (table 1). Research on CA1 proteome 7 days after stroke when broad cell death had occurred was useless to reveal the molecular mechanisms before death. We chose 24hour after stroke when injury effect on neuron had begun before their death as the time point versus sham control of observation. Brain coronal sections were acutely obtained from sham control animals, followed by dissociation of hippocampal subregions CA1 and CA3. Tissue was prepared before two dimensional electrophoresis, gel staining and image scanning to obtain gel images with good separation and reproducibility. Differentially displayed proteins between sham control CA1 and CA3 were revealed by analysis of professional software. 8 of those 9 picked protein spots were successfully sequenced by MALDI-TOF/TOF-MS. Proteins differentially expressed between the sham control and 24hr stroke groups were also uncovered in the same way, and 21 of those 23 picked protein spots were successfully sequenced. Hippocampal mitochondria were separated from rat of sham control group and 24h after stroke group, and then examined by electron microscopy. The mitochondria were fixed, sliced, stained and observed, A large proportion of (70-80%) extracts were mitochondria with intact membrane and cristae. Mitochondrial proteins differentially expressed between the sham control and 24hr stroke groups were also uncovered in the same way, except for MALDI-TOF-MS instead of MALDI-TOF/TOF-MS, and 13 of those 24 picked protein spots were successfully sequenced. Among the sequenced mitochondrial proteins, prohibitin was up regulated after stroke and validated by immunoblot to scale its dynamics (1, 6, 12, 24hour after stroke). Immunoblot results shew that accumulation of mitochondrial prohibitin didn't change lhour after stroke, but was up regulated 6hours after stroke (one-ANOVA, p p＜0.05, n=3).
The present study mined according to uniquely area specific susceptibility and key role of mitochondria in cell injury for molecular targets involved in stroke, mainly based upon the proteomic technologies of 2-DE tandem MS-TOF(/TOF)-MS. We found difference of proteome between sham control CA1 and CA3. The 8 successfully identified proteins were GFAP d (glial fibrillary acidic protein delta), Tpil (triose phosphate isomerase 1), aplysia ras-related homolog A2, rCRMP-3 (rat Collapsin response mediator proteins-3), HMGB1 (High mobility group box 1), platelet-activating factor acetylhydrolase isoform lb alpha2, protein similar to biliverdin reductase, glutamine synthetase 1。Glutamine synthetase 1 was more highly expressed in CA3, the others were more in CA1. rCRMP-3 may be a marker of postischemic neurogenesis. Tpil may contribute to higher susceptibility of CA1 for its higher expression in this area. Glutamine synthetase 1 can reasonably eliminate excess neurotoxic glutamate in CA3 to protect this area from stroke injury. We found differentially expressed proteins in CA1 after 24 hours of brain ischemia-reperfusion injury. The successfully identified proteins included down regulated unnamed protein product (HSP60 family), CKB (creatine kinase B), LASPI(LIM and SH3 protein 1), ATP synthase, brain abundant, membrane attached signal protein 1, protein similar to biliverdin reductase, and up regulated PGAM1 (phosphoglycerate mutase 1), stress-induced-phosphoprotein 1 (Hsp70/Hsp90-organizing protein), alpha B-crystallin, and DJ-1 protein. Creatine kinase B was not reported to be involved in ischemic area. We speculate that its role as a metabolic enzyme contribute to energy deficiency there. ATP synthase is the core enzyme to synthesize ATP, so its down regulation reasonably causes energy deficiency in ischemic area. Upregulation of PGAM1 indicated the increased glycolysis or energy deficiency in ischemic area; itself can be a potential therapeutic target. Whether the antiapoptotic role of alpha B-crystallin in myocarcial ischemia is also present in brain requires further investigation. Serum DJ-1 has already been considered as early marker of brain ischemia stroke, although its detailed local function in ischemic area remain unclear. We found differentially expressed proteins in CA3 after 24 hours of brain ischemia-reperfusion injury. The successfully identified proteins included down regulated nucleoside diphosphate kinase and upregulated voltage-dependent anion channel-like protein, brain creatine kinase, guanine deaminase, SPNA2 (brain Spectrin alpha chain), alpha synuclein, ATP synthase beta subunit, Rat Liver F1-Atpase Chain B. VDAC is included in the PTPC (permeability transition pore complex) which is proved to allow some proapoptotic components to release, so VDAC may have some effect on progress of brain ischemia. There was no report about role of nucleoside diphosphate kinase and so on in brain ischemia as much as we know. Some mitochondrial proteins in hippocampus were found to be differentially expressed after 24 hours of brain ischemia-reperfusion injury. The successfully identified proteins included down regulated HCNP (Hippocampal cholinergic neurostimulating peptide), PDHE1-A type I (Pyruvate dehydrogenase E1 component alpha subunit), LASP-1 (LIM and SH3 domain protein 1), ATP synthase beta subunit, Alpha-internexin, GSTp (Glutathione S-transferase pi), GMF-beta (Glia maturation factor beta), and upregulated Heat shock 70 kDa protein 1A/1B, Alpha-tubulin 3, Prohibitin, Synapsin-2, Heat shock cognate 71 kDa protein, and Guanine nucleotide-binding protein subunit beta 1. PDH (Pyruvate dehydrogenase) is key enzyme in TCA cycle (tricarboxylic acid cycle). Downregulation of PDH and ATP synthase would centainly interrupt the energy supply. GSTp previously implicated as stroke related gene's product may play protective role through inhibition of JNK and STAT pathways. Preceding research on prohibitin was primarily settled in productive organs and their derived carcinomas. Prohibitin was proved to be a key component of Rb-E2F pathway which plays the central role in mitosis. Recent studies have proved importance of aberrant cell cycle reentry before various nervous system diseases. Prohibitin has been shown to be involved in oxidative stress injury, which may due to its shuttle between mitochondria and nuclei. Our research can provided a new proof of crosstalk between those two organelles by prohibitin and its role in ischemic hypoxic brain diseases such as ischemic stroke. To verify the above hypothesis, we need to interfere the cellular expression and mitochondrial accumulation of prohibitin, and observe the survival of neurons and expression and activity of prohibitin related signal molecules
引文
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