子痫前期关键蛋白电化学生物传感器的研究和初步临床应用

发布时间：2018-09-17 13:27

【摘要】：子痫前期(preeclampsia, PE)作为一种妊娠期特发疾病,是导致孕产妇和围产儿发病率及死亡率上升的主要原因。子痫前期的发病机制极为复杂,尽管多年来对子痫前期发病机制的探讨一直是产科研究领域研究的热点和焦点,但迄今为止,仍无一种理论可以全面满意地阐述其病因。因此,临床治疗只能采取解痉、降压等对症治疗措施,疗效及预后并不理想。虽然目前关于子痫前期发病机制的基础研究达到了前所未有的水平,但仍缺乏准确、特异有效的临床预测指标,鲜有可单独用于临床的标志物。另一方面,近年来,生物医学研究正经历从定性到定量、从静态到动态、从分析到综合的阶段,迈向更高层次,对临床指标,譬如疾病相关蛋白质的分析检测也提出了更高更新的要求。发展简单、快速、灵敏、准确、特异,甚至动态、实时、原位的蛋白质分析检测方法,在蛋白质科学研究和疾病的诊断领域具有十分重要的意义。因此本论文基于蛋白质电化学生物传感技术,使用新型生物及化学探针,结合界面以及纳米科学等相关领域的新原理新技术,开展蛋白质的分子标记、分子识别、界面组装以及信号检测等方面的研究工作,实现能够对那些在未来极具潜力成为子痫前期发生发展预测评估指标的子痫前期关键蛋白,进行简单、快速、灵敏、准确、特异的检测分析的愿景。第一部分：基于锆离子信号放大检测CREB蛋白磷酸化水平的新方法目的：蛋白质的磷酸化修饰是基本的生物学现象,参与调控生物体内的许多生理过程。CREB (cAMP反应元件结合蛋白)作为一种至关重要的核转录因子,它的功能表现在了包括基因转录调节、细胞凋亡调控、免疫应答等生命活动的诸多方面,而CREB蛋白的生物学活性正是受其自身磷酸化水平所调控的。近年来的研究表明,CREB蛋白磷酸化水平的异常表达与子痫前期等许多疾病的发生发展密切相关。因此本章节构建了一种基于锆离子信号放大检测CREB蛋白磷酸化水平的新型电化学检测方法,并实现了该检测手段在临床实际样本中的应用。方法：以金纳米颗粒/DNA/亚甲基蓝纳米复合材料(GNP/DNA/MB)作为信号标记,通过Zr4+所介导的纳米复合信标对磷酸化位点的特异性标记,实现对CREB磷酸化水平的定量检测。在本工作中,首先将含有CREB蛋白特异性、高亲和力结合位点的捕获探针DNA序列修饰到金电极的表面,当这些探针捕获有磷酸化的CREB之后,利用Zr4+对磷酸基团的分子识别机制,可以将电极上捕获的磷酸化CREB蛋白与同样修饰有磷酸基团的GNP/DNA/MB相连接。本方法对纯品磷酸化CREB蛋白的检测限可低至0.25 nM。为了进一步验证该方法在实际生物样本中的检测能力,我们选取了南京医科大学第一附属医院产科住院分娩的正常孕妇、轻度子痫前期孕妇、重度子痫前期孕妇各5例,对孕妇胎盘组织中CREB蛋白的磷酸化水平进行了检测分析。结果：1.条件的优化：(1) 500unit/mL为PKA的最优浓度条件。(2)120分钟的孵育时间已经可以确保磷酸化CREB蛋白充分地结合到修饰有捕获探针的电极表面。(3)Zr4+溶液的浓度达到0.2mMM时,已经足以保证本实验体系信号的级联放大效果。(4)对于GNP/DNA/MB纳米复合信标与捕获有靶标蛋白的电极间的孵育时间,60分钟为最佳。2.通过本方法可获得CREB蛋白磷酸化水平的饱和增长曲线,并可据此建立标准工作曲线,用于复杂样品磷酸化水平的标定。3.在孕妇胎盘组织标本中,患有子痫前期的孕妇CREB蛋白磷酸化水平与正常孕妇相比,有所升高。随着疾病严重程度的增加,CREB磷酸化水平也逐渐升高。结论：该部分设计的CREB蛋白磷酸化水平电化学分析方案展示了理想的灵敏度,以及较高的特异性和良好的重复性。未来在临床实际样本中,在对生理状态和病理状态下的蛋白质磷酸化水平进行分析研究方面,具有较为广阔的应用前景。第二部分：基于多肽调控的蛋白质/DNA可逆相互作用检测STAT 3蛋白活化水平的新方法目的：信号转导和转录激活因子3(signal transducer and activator of transcription 3, STAT 3)参与调控了细胞的生长、分化、代谢及凋亡、血管生成等诸多生命活动。近年来的研究表明,STAT3蛋白活化水平的异常不仅与肿瘤的增殖、分化,血管生成,侵袭转移和免疫逃逸等生理功能高度相关,还在子痫前期等一系列妊娠相关疾病的发病机制中发挥着至关重要的作用。因此在本章节中,我们构建了一种基于多肽介导的STAT 3蛋白和DNA探针间可逆动态相互作用从而对STAT 3蛋白活化水平进行检测分析的电化学分析测试新方法,并将该方法成功运用于实际生物样本中。方法：本方案借助STAT3蛋白与特异性磷酸化多肽、DNA探针这两种天然配体间具有强度差异性的相互作用,引入功能化的纳米石墨烯复合信标作为信号放大的工具,实现了对STAT3蛋白活化水平的检测。在本工作中,首先将能够和STAT 3靶蛋白高特异性、高亲和力结合的末端修饰有磷酸基团的捕获探针双链DNA修饰在金电极表面。当电极上DNA探针捕获活化的STAT 3二聚体后,用DNase I内切酶降解电极上过量的未与STAT3结合的DNA探针,再利用特异性磷酸化多肽来促使靶标蛋白STAT 3二聚体的解离,从而使STAT 3与DNA分离。之后,借助Zr4+对磷酸基团的分子识别机制,将磷酸化纳米石墨烯复合信标固定到电极表面作为信号放大工具,保证了本方法可以实现对活化的STAT 3蛋白进行高灵敏的检测,检测限低至0.085 nM。为了进一步验证本方法在实际生物样本中的检测能力,我们选取了南京医科大学第一附属医院产科住院分娩的正常孕妇、轻度子痫前期孕妇、重度子痫前期孕妇各5例,对三组孕妇胎盘组织中STAT 3蛋白的活化水平进行了检测分析。结果：1.条件的优化：(1)120分钟的温育时间已经可以确保活化的STAT3蛋白充分地结合到修饰有捕获探针的电极表面。(2) DNase I最佳酶切反应时间为30分钟。(3)捕获有靶标蛋白的电极与磷酸化肽间的孵育时间为60分钟时,可以确保将电极上结合的STAT 3充分解聚洗脱。(4)对于磷酸化纳米石墨烯复合信标与重新释放了捕获探针的电极间的孵育时间,60分钟为最佳。2.在本体系中,STAT 3活化水平随活化的STAT 3蛋白浓度的增加而增高,到达一定浓度后饱和。3.在孕妇胎盘组织标本中,患有子痫前期的孕妇STAT 3蛋白活化水平与正常孕妇相比,有所降低。随着疾病严重程度的增加,STAT 3活化水平则逐渐降低。结论：此部分设计的STAT 3蛋白活化水平电化学分析方案表现出理想的灵敏度,较高的特异性和良好的重复性。未来在临床复杂样本中,在对生理状态和病理状态下的蛋白质活化水平进行定量分析研究方面,具有着重要的生物学意义和广阔的应用前景。第三部分：借助超分子识别对Corin酶进行低背景噪音、灵敏检测的新方法目的：蛋白酶切是蛋白质翻译后修饰的重要手段之一。丝氨酸蛋白酶作为Ⅱ型跨膜嵌合蛋白酶超家族中的重要一员,在限制性蛋白水解过程中起重要作用,参与调控体内激素成熟、细胞凋亡、血压调节、血液凝固等众多的生理过程。近年来的研究表明,Corin作为一种新近发现的Ⅱ型跨膜丝氨酸蛋白酶,其酶活性水平的异常不仅与高血压、病理性的心肌肥厚、心力衰竭、肿瘤等多种疾病的发生发展高度相关,还在子痫前期的发病机制中发挥着至关重要的作用,极具潜力成为可预测、评估子痫前期发生发展过程的临床分子标志物。因此发展简单、快速、灵敏、准确、特异的蛋白酶活性检测方法,不仅可以为相关疾病的病理研究提供新的思路和研究平台,还在临床诊断、个体化治疗、药物筛选、靶向治疗等应用方面具有巨大的潜力。方法：本研究以一段既具备Corin特异性水解底物序列,又具有葫芦脲识别位点的双功能多肽作为探针,首先使探针在电极表面形成传感阵列,在没有Corin的情况下,电极表面未被切割的多肽探针可以提供葫芦脲识别位点,利用两个处于氨基末端的苯丙氨酸可被一个八聚葫芦脲包裹形成超分子复合物的特性,通过多肽探针、滚环扩增引物DNA和葫芦脲间特异性的超分子识别机制,将引物DNA固定到电极表面并诱导电极界面的滚环扩增反应。相反,在有Corin作用下,多肽探针会在特异性位点被水解,带有葫芦脲识别位点的多肽片段会从电极表面脱离,电极表面无法形成超分子复合物,因而引物DNA不能被引入到电极界面,无法诱导滚环扩增反应。由于滚环扩增产生的长链DNA产物对电极表面有很强的封闭作用,因此,借助于这种封闭作用的有、无可极大地放大有Corin是否存在情况下的信号增量,且信号增量与Corin酶活性的大小呈正相关,因而实现了对Corin酶活性水平低背景信号、高特异性和高灵敏度的检测。为了进一步验证本方法在实际生物样本中的适应性,我们选取了南京医科大学第一附属医院产科住院分娩的正常孕妇、轻度子痫前期孕妇、重度子痫前期孕妇各5例,对三组孕妇血浆组织中Corin的酶活性水平进行了检测分析。结果：1.条件的优化：(1)16小时的温育时间已经足以保证多肽探针、滚环扩增引物DNA和葫芦脲间特异性的超分子识别,可确保电极表面超分子复合物的形成。(2) Corin对电极上修饰的多肽探针最佳酶切反应时间为30分钟。(3)电极上滚环扩增最佳反应时间为60分钟。2.在本体系中,电化学信号响应随着Corin酶活性水平的升高而增高,到达一定浓度后信号响应饱和。3.在孕妇血浆标本中,患有子痫前期的孕妇Corin酶活性水平与正常孕妇相比明显升高。随着疾病严重程度的增加,血浆中的Corin酶活性水平也逐步升高。结论：该部分设计的Corin酶活电化学分析方案展示了理想的灵敏度,超低的背景信号,高度的特异性和良好的重复性。未来在临床复杂生物样本中,在对生理状态和病理状态下的蛋白酶活性水平进行定量分析研究方面,可以提供优良的检测分析手段,具有广阔的应用前景。
[Abstract]:Preeclampsia (PE), as a special disease of pregnancy, is the main cause of increasing morbidity and mortality of pregnant women and perinatal infants. No theory can fully and satisfactorily explain the etiology of preeclampsia. Therefore, clinical treatment can only take symptomatic measures such as spasmolysis, hypotension and so on. The curative effect and prognosis are not ideal. Although the basic research on the pathogenesis of preeclampsia has reached an unprecedented level, there is still a lack of accurate, specific and effective clinical predictive indicators. On the other hand, in recent years, biomedical research is experiencing from qualitative to quantitative, from static to dynamic, from analysis to comprehensive stage, to a higher level. The analysis and detection of clinical indicators, such as disease-related proteins, also put forward higher and newer requirements. Up to now, real-time, in-situ protein analysis and detection methods are of great significance in the field of protein science research and disease diagnosis. Therefore, based on protein electrochemical biosensor technology, this paper uses new biological and chemical probes, and combines new principles and technologies in the fields of interface and nanoscience to develop eggs. Molecular markers, molecular recognition, interfacial assembly and signal detection of white matter have been studied to realize the prospect of simple, rapid, sensitive, accurate and specific detection and analysis of key proteins in preeclampsia that have great potential to be used as predictors of the development of preeclampsia in the future. Ion signal amplification is a new method to detect the phosphorylation level of CREB proteins. Protein phosphorylation is a basic biological phenomenon and involves in regulating many physiological processes in organisms. Recent studies have shown that the abnormal expression of CREB protein phosphorylation is closely related to the occurrence and development of many diseases such as preeclampsia. A novel electrochemical method for detecting the phosphorylation level of CREB protein by zirconium ion amplification was developed, and its application in clinical samples was realized. METHODS: The phosphorylation sites of gold nanoparticles / DNA / methylene blue nanocomposites (GNP / DNA / MB) were detected by Zr4 + mediated nanocomposite beacons. In this work, the DNA sequence of capture probes containing CREB protein-specific and high affinity binding sites was first modified to the surface of gold electrode. After these probes capture phosphorylated CREB, the phosphorylated CREB could be identified by Zr4+ molecular recognition mechanism. The captured phosphorylated CREB protein was linked to GNP/DNA/MB, which was also modified with phosphorylated groups. The detection limit of phosphorylated CREB protein could be as low as 0.25 nM. In order to further verify the detection ability of this method in real biological samples, we selected normal pregnancies of obstetrics in the First Affiliated Hospital of Nanjing Medical University. The phosphorylation level of CREB protein in placenta of pregnant women, mild preeclampsia pregnant women and severe preeclampsia pregnant women were detected and analyzed. Results: 1. Optimization of the conditions: (1) 500 unit/mL is the optimal concentration of PKA. (2) 120 minutes incubation time can ensure that phosphorylated CREB protein is fully bound to the modified protein. (4) The incubation time between GNP/DNA/MB nanocomposite beacon and the electrode capturing the target protein is the best. 2. The saturated increase of phosphorylation level of CREB protein can be obtained by this method. The phosphorylation level of CREB protein in pregnant women with preeclampsia was higher than that in normal pregnant women. The phosphorylation level of CREB protein in pregnant women with preeclampsia increased gradually with the severity of the disease. The electrochemical assay of phosphorylation level of CREB protein demonstrated ideal sensitivity, high specificity and good repeatability. In the future, it will be widely used in the analysis and research of phosphorylation level of protein in physiological and pathological states in clinical samples. A novel method for detecting STAT 3 protein activation based on protein/DNA reversible interactions regulated by peptides Objective: Signal transducer and activator of transcription 3 (STAT 3) is involved in regulating cell growth, differentiation, metabolism, apoptosis and angiogenesis. Studies have shown that the abnormal activation of STAT3 protein is not only highly correlated with tumor proliferation, differentiation, angiogenesis, invasion, metastasis and immune escape, but also plays an important role in the pathogenesis of preeclampsia and other pregnancy-related diseases. Mediated reversible dynamic interaction between STAT3 protein and DNA probe, a new electrochemical assay method was developed to detect and analyze the activation level of STAT3 protein. The method was successfully applied to practical biological samples. In order to detect the activation level of STAT3 protein, functional graphene nanocomposite beacon was introduced as a signal amplification tool. In this work, the double-stranded DNA, which can bind to STAT3 target protein with high specificity and affinity, was modified into gold electrodes. Pole surface. When DNA probe on the electrode captures the activated STAT 3 dimer, the excessive DNA probe not bound to STAT 3 on the electrode is degraded by DNase I endonuclease, and then the target protein STAT 3 dimer is disintegrated by specific phosphorylated peptides to separate STAT 3 from DNA. The phosphorylated nano-graphene composite beacon was fixed on the electrode surface as a signal amplification tool, which ensured that the method could be used to detect the activated STAT-3 protein with high sensitivity. The detection limit was as low as 0.085 nM. In order to further verify the detection ability of the method in the actual biological samples, we selected the first Nanjing Medical University. The activation levels of STAT 3 protein in placenta of normal pregnant women, mild preeclampsia pregnant women and severe preeclampsia pregnant women were detected and analyzed. Results: 1. Optimization of the conditions: (1) 120 minutes of incubation time can ensure that the activated STAT 3 protein is fully bound to repair. The optimal digestion time of DNase I was 30 minutes. (3) When the incubation time between the electrode and the phosphopeptides was 60 minutes, the STAT3 bound to the electrode could be fully depolymerized and eluted. (4) The phosphorylated nanographene composite beacon and the capture probe were re-released. In this system, STAT 3 activation level increased with the increase of STAT 3 protein concentration, reached a certain concentration and saturated. 3. In pregnant women placenta tissue samples, preeclampsia pregnant women with STAT 3 protein activation level decreased compared with normal pregnant women. CONCLUSION: The electrochemical assay of STAT 3 protein activation level designed in this part shows ideal sensitivity, high specificity and good reproducibility. Part 3: A new method for detecting Orin enzyme with low background noise by supramolecular recognition. Purpose: Protease digestion is one of the important means for post-translational modification of proteins. Serine proteases are the heavy proteins in type II transmembrane chimeric protease superfamily. In recent years, studies have shown that Corin, as a newly discovered type II transmembrane serine protease, has abnormal enzyme activity levels not only in hypertension but also in pathology. Myocardial hypertrophy, heart failure, tumor and other diseases are highly related to the occurrence and development of preeclampsia, but also play a vital role in the pathogenesis of preeclampsia, has great potential to be predictable, assessment of the occurrence and development of preeclampsia clinical molecular markers. The assay method can not only provide a new idea and research platform for pathological study of related diseases, but also have great potential in clinical diagnosis, individualized treatment, drug screening, targeted therapy and other applications. Peptides are used as probes to form sensor arrays on the electrode surface. Without Corin, the uncut polypeptide probes on the electrode surface can provide cucurbitacin recognition sites. Two phenylalanine at the amino terminal can be encapsulated by an octagourea to form a supramolecular complex. Through the polypeptide probes, the cucurbitacin recognition sites can be rolled. Cyclic amplification primer DNA and cucurbit urea specific supramolecular recognition mechanism, primer DNA is fixed to the electrode surface and induce the roll-around amplification reaction at the electrode interface. Conversely, under the action of Corin, the peptide probe will be hydrolyzed at the specific site, the polypeptide fragment with cucurbit urea recognition site will detach from the electrode surface, and the electrode surface can not. As a result, primer DNA can not be introduced into the electrode interface and can not induce ring-rolling amplification reaction. Because the long-chain DNA products produced by ring-rolling amplification have a strong sealing effect on the electrode surface, whether the signal increment in the presence or absence of Corin can be greatly amplified by means of this sealing effect. In order to further verify the applicability of this method in biological samples, we selected normal pregnant women who delivered in the obstetrics department of the First Affiliated Hospital of Nanjing Medical University. Corin activity in plasma of preeclampsia pregnant women and severe preeclampsia pregnant women were measured and analyzed. Results: 1. The optimum conditions were as follows: (1) 16 hours of incubation time was enough to ensure the specific supramolecular recognition between polypeptide probe, roll-ring amplification primer DNA and cucurbitacin, which could ensure the electrode surface. The formation of supramolecular complexes. (2) The optimal enzyme digestion time of Corin on the electrode modified polypeptide probe was 30 minutes. (3) The optimal reaction time of roll-ring amplification on the electrode was 60 minutes.2. In this system, the electrochemical signal response increased with the increase of Corin enzyme activity, and saturated after reaching a certain concentration. Corin enzyme activity in plasma samples of preeclampsia pregnant women was significantly higher than that of normal pregnant women.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714.244
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本文编号：2246058