【作者】 张更谦；

【导师】 张林；

【作者基本信息】 四川大学 ， 法医学， 2007， 博士

【摘要】 第一部分SELDI-TOF MS筛选大鼠急性心肌缺血早期标记物的研究背景冠心病在我国的发病率逐年上升，病死率和致残率居高不下，也是构成法医学猝死的主要原因。冠心病病人约有1／4无明显的胸痛等症状，约50％无心电图改变。法医学实践中也发现：如果心肌缺血发生在室间隔等重要部位，即使是微小的心肌梗死，也会引起心源性猝死。临床上常用肌钙蛋白等作冠心病的辅助诊断，但该蛋白是确诊心肌梗死的指标，在血浆中要2-4 h以后才出现，更早的心肌缺血指标如C反应蛋白，心肌脂肪酸结合蛋白等特异性较差，需要和肌钙蛋白联合应用，检测方法繁复。因此，寻找新的特异性好的心肌缺血的早期标志物显得尤为重要。SELDI-TOF MS是近年来新的蛋白质组分析工具，其原理是：用不同性质的化学芯片结合不同性质的蛋白质和多肽，经过能量分子结合后受激光的激发进入质谱，以其在质谱中的不同飞行时间来分离蛋白。该方法广泛用于寻找疾病蛋白标记。我们采用了SELDI-TOF MS的观察大鼠急性心肌缺血后的血清蛋白变化规律，寻找能作为心肌缺血的蛋白标记。同时我们也观察了心肌本身蛋白质谱的改变。方法SD大鼠分成手术组，假手术组和非手术对照组。在结扎大鼠冠脉前降支，在5 min-6 h内不同的时间段取大鼠的静脉血和心肌组织。血清和心肌组织的蛋白提取物分别经过化学芯片的初步筛选后均选定CM10芯片作为实验用芯片。用Ciphergen公司的PBSⅡ-C型SELDI-TOF MS质谱仪检测。同时血清进行了cTnI和CKMB的检测，心肌进行常规HE染色。结果只出现于手术组而几乎不出现于假手术组和非手术对照组的峰为特异峰；出现于手术组和假手术组而在非手术组中不出现的峰称非特异峰。血清CM10芯片检测到3个心肌缺血特异性峰，质荷比(m／z)分别是7564 Da、7586 Da、9583 Da。其中7564 Da、7586 Da相伴出现，在缺血5min组阳性率50％，在15 min以后各缺血组的阳性率达到97％，在假手术组仅有1个样本有这两个峰，未见于非手术组，特异性达到97％。9583 Da仅存在于缺血后1 h以内。发现价在手术组和假手术组都出现的非心肌特异性峰(4983 Da、5140 Da、8075 Da、9423 Da)。4个峰值改变的非特异性峰：3个升高的峰(4400 Da、4542 Da、8400 Da)，1个在假手术组和手术组都降低的峰(7666 Da)。缺血心肌组织同两个对照组比较，主要有两种差别峰：特异性出现的新峰和峰值升高的峰。特异性出现的新峰3个：质荷比(m／z)分别为6304Da、8337 Da、8376 Da，这三个峰在手术组中阳性率为100％，假手术组和非手术组中均没有发现，其中峰6304 Da，8337 Da在手术5 min后就升高，8376 Da也是在缺血5 min后出现，15 min后峰值明显升高。特异性升高的峰3个：其中质荷比(m／z)为6658 Da、6876 Da两个峰都是在实验组中15 min以后升高2倍以上，8577 Da峰则在缺血5 min以后升高。cTnI和CKMB在血清中的含量分别均在4 h以后升高。HE染色在1 h可见心肌收缩带，4-6 h可见有炎症细胞浸润。结论血清中出现7564 Da、7586 Da蛋白峰，缺血心肌组织中出现6304Da、8337 Da、8376 Da蛋白峰都是急性心肌缺血后的特异性改变，是目前已知检测出的最早的蛋白标记物。有望用于临床心肌缺血的早期诊断和法医学心源性猝死的诊断。第二部分大鼠急性心肌缺血后差异基因的筛选背景了解心肌缺血后早期分子变化的机制，对于寻找疾病的标记和干预治疗缺血性心肌病意义重大。目前关于心肌缺血在转录水平上的研究多采用cDNA芯片的方法，该方法用已知的EST和基因做探针，其结果取决于芯片上探针的密度，而且对于低表达的RNA效果差，更无法了解可能出现的新基因。抑制消减杂交(SSH)是一种从转录水平比较两种材料mRNA差异的方法，其设计方法从一定程度上弥补了DNA芯片的不足。我们用该方法检测了大鼠心肌缺血后的差异表达基因。方法8只SD大鼠心肌缺血1 h后处死，提取纯化mRNA，以缺血心肌作为Tester，非缺血自身对照作为Driver，进行正向杂交，以非缺血心肌作为Tester，缺血心肌作为Driver进行反向杂交。扩增产物构建消减cDNA文库，经过测序和斑点杂交的确认后，得到不同基因的克隆片断，并对测序结果进行生物学分析。结果正向杂交得到的已知基因有16个：分为以下五类：(1)心肌和细胞骨架：myoglobin，troponin T2，cardiac(Tnnt2)，tropomyosin 1 alpha(Tpm1)，Rattus norvegicus beta-glo(MGC72973)。(2)蛋白翻译相关：ribosomal protein L39，ribosomal protein S11(Rps11)，eukaryotic translation elongation factor 1 alpha 1(Eef1a1)。(3)代谢以及相关酶类：ATPase 3(Psmc3)，malate dehydrogenase 1 NAD，lactate dehydrogenase B(Ldhb)，UDP-glucose pyrophosphorylase 2(Ugp2)。(4)心肌重塑相关：cathepsin L(Ctsl)。(5)其他：Sjogren syndrome antigen B(Ssb)，basigin(Bsg)，unc-45 homolog A(Unc45a)。反向杂交的到的已知基因有1个氧化还原相关蛋白NADH dehydrogenase(ubiquinone)Fe-S protein 2(Ndufs2)。共获得新克隆16个，其中9个可以在REFRNA库中查到并可根据NCBI的DNA数据库预测其功能：7个核糖体相关，一个为Telethonin，一个为actin。另外7个完全未知，无法根据DNA数据库预测其可能序列和功能。其中Sjogren syndrome antigen B(Ssb)，basigin(Bsg)，unc-45 homolog A(Unc45a)，cathepsin L(Ctsl)，Telethonin均为第一次发现与早期心肌缺血有关。其功能尚需进一步证实。结论我们构建了大鼠心肌缺血后的消减杂交文库。五个基因第一次报道与心肌缺血有关，分别是Sjogren syndrome antigen B(Ssb)，basigin(Bsg)，unc-45 homolog A(Unc45a)，cathepsin L(Ctsl)和Telethonin，发现了7个新的EST与心肌缺血相关的序列。更多还原

【Abstract】 Chapter 1 Potential biomarkers discorvery of acute myocardial ischemia using SELDI-TOF MSBackground The increasing prevalence of Coronary Artery Disease (CAD) has high mortality and morbidity. Besides, CAD constructs the major death cause of sudden death in forensic science. About 1/4 CAD patients have not pectoral syndrome and half of them do not show electrocardiogram changes. Forensic pathologist found that when the important area is ischemia, such as ischemia of ventricular septal, could lead to sudden cardiac death (SCD). Troponin (cTn) is widely used as ancillary clinical diagnosis of CAD. As a marker of myocardial infarction, cTn could be detected 2-4 h in the plasma after the onset of the clinical syndrome. The earlier biomarkers, such as CRP and HABP, had to be used for earlier diagnose of cardiac ischemia for their poor specificity. SELDI-TOF MS was novel proteomic tool. According to its principle, the proteins were captured by ProteinChips which can bind with different proteins or peptides for their different chemistry properties, after that, the proteins or peptides were bound with energy molecules and run in the mass spectrometry when inspired by laser. The peptides were separated by their flying time in the MS. This equipment was widely used to find disease biomarkers. In this study, SELDI TOF MS were used to detect the pepitides profile and biomarkers of cardiac ischemia in the sera and myocardium.Methods Sprague-Dawley rats were divided as three groups: operation group, sham operation group, non-operation group. The sera and ischemia myocardium were collected 5 min-6 h after the coronary artery ligation. CM10 ProteinChip was selected as the experiment chip. The sera and myocardium protein extracts were processed by CM10 ProteinChip and analyzed by SELDI-TOF MS. CKMB, cTnI and histological change were detected too.Result The peaks detected in the operation groups and nearly seen in the sham operation groups were called as specific peaks. Those peaks emerged in the operation groups and sham operation groups but not in the non-operation groups were called as non-specific groups. Three peaks with m/z 7564Da, 7586 Da and 9583 Da were only found in the operation groups’ sera. The peaks 7564Da and 7586 Da were seen simultaneously had the sensitivity of 97.0% and specificity of 97.0% in the>15 min operation groups. Some non-specific peaks in response to injury have also been seen, they are the peaks with the m/z 4983 Da, 5140 Da, 8075 Da, 9423 Da. Four non-specifc peaks with marked peak height changes: 4400 Da, 4542 Da, 8400 Da(increased), and 7666 Da(decreased). Two kinds of peaks were found in the myocardium protein extraction. One is the three novel specific peaks which were only found in operation groups with the 100% specificity: 6304Da, 8337Da, 8376Da. Peak 6304Da, 8337Da were found with a high peak height >5 min operation group, while peak height of 8376Da was low in 5 min group but increased in the >15min groups. Another kind of peaks was specific peaks with increased peak height. Peak 6658 Da, 6876 Da increased in>15min groups, peak 8577 Da increased in >5 min groups. While the significant increase of CKMB and cTnI in the serum could be detected until 4 hours after the ligation. For the HE staining of the myocardium, contraction band was observed after 1 h of the ligation, inflammatory cells were found in the ischemia area 4-6 h after the ligation.Conclusion Whether in the serum or the myocardium, the change peaks found by SELDI-TOF MS were the earliest changes after cardiac ischemia by now. These changes may reflect the modified peptides or novel proteion. They may be used as the biomarker of cardiac ischemia for clinical purpose or ancillary ischemia sigh for SCD.Chaper 2 Screening expreesed genes in a rat model during cardiac ischemia by suppression subtractive hybridizationBackground Understanding the molecular changes after the cardiac ischemia is pivotal for the diagnosis of myocardial infarction and therapy of the ischemia cardiac diseases. By now, The main approch used for research on myocardial ischemia transcription is microarray detection. This method can only detect the known genes and ESTs and limited by the molecule transcription level. Suppression Subtractive Hybridization (SSH) is a approch to detect the different transcription gene between two groups. The SSH is complementary to cDNA microaray. We detected the transcription changes after cardiac ischemia by SSH in this study.Method Eight Sprague-Dawley rats were sacrificed after the coronary artery ligation for 1 h and the myocardium were collected. After the mRNA was extracted, a PCR-Select cDNA Substraction Kit was used as the manufacture’s protocol. Two hybridizations were established: the forward hybridization: ischemia myocardium cDNA were used as the tester, and non-ischemia cDNA as the driver; the reverse hybridization: non-ischemia myocardium cDNA as the tester, and ischemia cDNA as the driver. The subtractive cDNA library were sequenced and identified by dot blot.Result Sixteen known genes were sequenced after forward hybridization: they can be classified as the following: (1) Myocardium gene: myoglobin, cardiac troponin T2, tropomyosin 1, Rattus norvegicus beta-glo. (2)Translation related gene: ribosomal protein L39, ribosomal protein S11 (Rps11),eukaryotic translation elongation factor 1 alpha 1. (3) Metabolism and enzymes: ATPase 3 (Psmc3), malate dehydrogenase 1, NAD, lactate dehydrogenase B (Ldhb), UDP-glucose pyrophosphorylase 2 (Ugp2).(4) Myocardium remodeling: cathepsin L (Cts1). (5) Others: Sjogren syndrome antigen B (Ssb), basigin (Bsg), unc-45 homolog A (Unc45a). Only one known gene, NADH dehydrogenase (ubiquinone) Fe-S protein 2, was found in the reverse hybridization. Sixteen unknown clones were detected: Nine of them can be aligned in the REFRNA databank, including 7 sequences which were predicted similar to ribosomal protein, and two others were telethonin and actin; Seven of them were totally unknown about there function. Among them, Ssb, basigin, unc-45 homolog A (Unc45a),cathepsin L (Cts1) and Telethonin were first reported to be associated with acute myocardial ischemia.Conclusion We established the SSH library of rat ischemia (ischemia duration for 1 h). Five genes including Ssb, basigin, unc-45 homolog A (Unc45a), cathepsin L (Cts1) and Telethonin were first found to be associated with acute myocardial ischemia. Seven totally new ESTs were found.更多还原