神经系统副肿瘤综合征的生物标志物检测
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摘要
肿瘤分为良性肿瘤和恶性肿瘤两种。一般来讲,后者就是我们常说的癌。近些年,伴随着环境问题、食品安全等问题的不断加剧,癌症的发病率以及死亡率也在逐年攀升。癌症,成为一种让人们谈虎色变的病症。副肿瘤综合征一般是指由于肿瘤的产物异常而引起的免疫反应或者一些其他的不明原因而造成的机体在神经、造血、消化、内分泌、骨关节、肾脏及皮肤等系统发生病变进而出现一些相应的临床表现的现象。由于这些病变一般不是由原发肿瘤直接引起,而是由以上途径间接引起的,故称为副肿瘤综合征。
神经系统副肿瘤综合征(paraneoplastic neurologic syndrome, NPS)是发生在某些恶性肿瘤患者体内,在肿瘤没有发生转移的情况下,而引起自身其它部分或者器官的神经系统病变。它既非肿瘤的直接浸润或者转移,也非代谢、营养、感染以及抗肿瘤治疗引起。其表现常为肌肉,周围神经,甚至脊髓或者脑的功能性障碍。神经系统综合征(PNS)在恶性肿瘤患者中发病率一般<1%,但是病变范围较广,临床上表现多样。神经系统副肿瘤综合征(PNS)明显的神经系统表现可以发生在患者恶性肿瘤发生之前,同时或者之后。但通常来讲,神经系统副肿瘤综合征(PNS)一般发生在患者肿瘤发生之前较多一些。据统计约有80%左右的神经系统副肿瘤综合征(PNS)患者在肿瘤发生之前就有了明显的神经系统病变表现。这就为我们及早的发现恶性肿瘤的存在提供了一个契机。对于严重威胁人类生命健康的癌症,如果能及早的发现以及诊断治疗,那么就可以显著地提高患者的生存期。
相对于一般的神经系统肿瘤症状来说,虽然神经系统副肿瘤综合症(PNS)的发病率较低,与其他系统肿瘤的神经系统非转移性并发症相比,副肿瘤综合征也很少见,但是因为种种因素,神经系统副肿瘤综合征(PNS)就临床上而言,及早的发现和治疗还是非常重要的。首先,神经系统副肿瘤综合征的出现会对神经系统的功能的缺损造成更加严重的伤害,并且病症的持续时间更加长久；其次,对于一般的神经系统病症患者出现的副肿瘤综合征现象,其往往是一种还没能够诊断出来的原位癌的特征,因此,这对于神经科医生而言,这是对患者能够及早的诊断病症的一种有效途径,同时,这也需要医生具有较高的敏锐的识别能力和诊断能力；再次,如果能够早期发现诊断并且治疗副肿瘤综合征,可以使肿瘤患者治愈的可能性大大提高,并且还可以逐步恢复神经系统功能。
目前来讲,神经系统副肿瘤综合征还没有特别有效的治疗方法。现在通常用维生素类的药物,皮质类固醇,血浆置换以及免疫抑制剂等方法进行治疗,但是疗效均未得到证实。据一些临床治疗结果表明,部分患者在对原发肿瘤进行有效治疗后,其神经系统副肿瘤综合征的症状得到明显的缓解和好转。
本研究的主要创新点：
1.本研究设计了一种新颖的量子点纳米粒子。直接用适体DNA作为水溶性纳米粒子反应制备过程中的稳定剂和参与反应的化合物,简化了一般实验需要单独将适体DNA修饰到纳米粒子表面上的繁琐步骤。
2.由于在单个量子点纳米粒子表面可以同时具有多个适体DNA,且在单个微球载体表面可以同时结合多个量子点纳米粒子,所以这具有一种放大作用,显著地提高了检测的效率和灵敏度。
第一部分：神经系统副肿瘤综合征的生物学标记物检测
目的：研究神经系统副肿瘤综合征(paraneoplastic neurologic syndrome, PNS)早期生物学标记物的检测,探讨其早期的诊断和早期的预防性治疗。
方法：
资料来源于2010年09月至2013年05月期间在我院病房及门诊就诊的患者32例,其中PNS患者12例,无PNS的其它疾患者20例。12例的PNS患者中男7例,女5例,年龄在35-72岁,平均年龄49.3±11.7岁,发病病程在3月-5年,其中小细胞肺癌(small-cell lung cancer,SCLC)5例,乳腺癌3例,卵巢癌2例,肝癌1例,嗜铬细胞瘤1例；20例的无NPS的其它疾患者中男12例,女8例,年龄在38-75岁,平均年龄50.5±9.1岁,发病病程在5月-4.5年,其中胃癌5例,系统性红斑狼疮5例,类风湿性关节炎3例,前列腺癌3例,皮肌炎2例,肺结核2例。所有诊断均经影像学、血液学相关检测及病理检查予以确认。另选取20例的同期健康体检者,其中男10例,女10例,年龄在26-65岁,平均年龄40.1±9.2岁。
对所有患者的年龄、性别、疾病病程、其它实验室检查(包括肌电图、脑电图等)、影像学特点(包括CT、M R I、增强M R I、MRA等)等因素进行分析评价。将所有的入选对象进行相关的临床检查及酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)定量检测患者血清的抗-Hu抗体、抗-Yo抗体、抗-Ri抗体。并将特异性抗体的检测的高低与神经系统副肿瘤综合征的原发病灶及临床表现进行比较及评价。
结果：
(1)两组患者的一般资料比较结果：
两组患者的病例数、年龄、性别、疾病病程等一般指标经t检验表明,两组间差异无统计学意义(P>0.05)。
(2) ELISA定量检测实验结果：
本研究ELISA结果显示PNS组抗-Hu抗体、抗-Ri抗体、抗-Yo抗体表达较非PNS组显著升高,统计有显著差异性(P<0.01)。
(3)特异性抗-Hu抗体在NPS诊断中的阳性预测值：
根据Graus等的NPS诊断标准,12例中9例确诊PNS或发现相关肿瘤,其中2例(见表1中的病例9和病例12)未发现原发肿瘤,另7例发现肿瘤。抗Hu抗体在NPS诊断中的阳性预测值为75%(9/12)。
(4)神经元抗体与NPS临床类型之间的关系：
抗-Hu抗体阳性的NPS患者主要表现为副肿瘤性感觉性神经元病(paraneoplastic sensory neuronopathy, PSN)和副肿瘤性脑脊髓炎(paraneoplastic encephalomyelitis, P EM),少数可表现为副肿瘤性边缘叶脑炎(paraneoplastic limbic encephalitis, PL E)、副肿瘤性小脑变性(paraneoplastic cerebellar degeneration, PCD)和副肿瘤性感觉运动性神经病(paraneoplastic sensory-motor neuropathy, PSMN)等；抗-Yo抗体主要导致PCD；抗-Ri抗体主要与副肿瘤性眼阵挛-肌阵挛(paraneoplastic opsoclonus-myoclonus, POM)有关。
结论：
1.神经系统副肿瘤综合征的主要生物学标记物为抗-Hu抗体、抗-Yo抗体、抗-Ri抗体三种抗体。
2. ELISA结果显示PNS组抗-Hu抗体、抗-Ri抗体、抗-Yo抗体表达较非PNS组显著升高,统计有显著差异性(P<0.01)。
3.检测患者血清的抗-Hu抗体、抗-Yo抗体、抗-Ri抗体,以此来提高肿瘤细胞早期检测,为临床的早期治疗提供诊断依据。
第二部分：基于量子点的电化学生物传感器检测肿瘤细胞及其标志物
目的：
通过改进的方法制备经适体DNA修饰过量子点纳米粒子和探针之后,采用阳极溶出伏安法对特定离子的检测以达到对目标肿瘤细胞以及肿瘤细胞表面的的标志物检测。进而为医学上成功检测肿瘤细胞提供一种可行的有效手段
方法：
硫化铅量子点和硫化镉量子点的制备是在参照了相关文献之后,对其合成工艺做了改进而得出的合成方法。首先将硝酸铅固体粉末和氯化镉固体粉末配制成一定浓度的溶液,然后依次加入巯基乙酸和连接DNA反应,半小时后再加入适量Na2S溶液反应24h便可得到相应的量子点纳米粒子。整个实验过程是在通氮气保护的条件下进行的,所得产物4℃保存备用。
探针的制备分为磁性探针和非磁性探针两种。磁性微球与非磁性聚苯乙烯微球在使用之前均用PBS缓冲溶液清洗。然后将事先准备好的MUC1适体溶液和事先制备的CdS量子点于磁珠体系当中,将HER-3适体溶液和事先制备的PbS量子点于聚苯乙烯微球体系当中,恒温孵育便可得到相应的纳米探针。
最终的电化学检测肿瘤细胞所采用的方法为阳极溶出伏安法(DPV)。用硝酸溶液将固载在肿瘤细胞表面的探针脱离下来并释放出Cd2+和pb2+。将事先准备好的玻碳电极至于电解池当中,然后取出电极并置于已经准备好的待测样品体系当中,开始进行差分脉冲伏安扫描,设置静置时间为15秒,脉冲增幅50mV,脉冲宽度50ms,电位扫描范围为-0.30to-0.80V(vs. Ag/AgCl)。在电位-0.65V和-0.45V所出现的阳极溶出峰值电流分别为Cd2+和pb2+的阳极溶出信号。
结果：
通过对实验条件的不断优化最终得到硫化铅量子点和硫化镉量子点这两种量子点的最佳制备条件。合成CdS量子点的最优条件为10mL浓度为1.0×10-3M的氯化镉溶液,10mL浓度为1.34×10-3硫化钠溶液,稳定剂为1μL基乙酸(C2H4O2S)和300μL1.0×10-6M连接DNA；合成PbS量子点的最优条件为10mL浓度为2×10-4M的硝酸铅溶液,4mL浓度为6.7×10-4M硫化钠溶液,稳定剂为1μL巯基乙酸(C2H4O2S)和300μL1.0×10-6M连接DNA。
通过对实验条件的不断优化最终得到在电化学检测过程中的最佳检测条件为：适体DNA的最佳浓度为4.0×10-7M；最佳pH值为4.7；探针制备孵育时间为30h。
最终对肿瘤细胞的检测,在细胞浓度1.0×102cells mL-1—1.0×106cells mL-1的变化范围内,电化学检测信号与细胞浓度的对数值成正比例关系,其校正曲线为I(μA)=-14.0024+9.7932lgC(cell mL-1),其中I为扣除空白电流之后的电流强度,C为细胞液中细胞的浓度,线性相关系数为0.995,n=9。进一步计算得到其检测限为31.7cells mL-1；单个靶细胞表面上的MUC1目标蛋白的数量约为6.41×104个,单个靶细胞表面上的HER-3目标蛋白的数量约为3.47x104个。同时,这种检测方法经实验证实在混合样品甚至更加复杂的环境中依然可以达到很好的检测效果。
结论：
本实验提出了一种全新的检测肿瘤细胞以及肿瘤标记物的方法。通过对生物传感器的设置,将量子点与生物传感器相结合,最后再固载至磁珠和聚苯乙烯微球这两种载体上,制成了一种基于CdS和PbS两种量子点的新型的电化学生物传感器。再将生物传感器与适体DNA相结合,这就赋予了生物传感器对特定目标的专一性。本实验中,我们在生物传感器上连接了MUC1和HER-3这两种适体,用于分析检测人类乳腺癌细胞(MCF-7)。
利用连接DNA作为合成量子点的稳定剂是本实验的一大特色,因为有它的存在才使得将量子点固载至磁珠或者聚苯乙烯微球上成为可能。然后将两种适体DNA分别连接到经量子点修饰过的磁珠和聚苯乙烯微球表面制成纳米探针。然后通过适体对目标蛋白的特异性识别是的探针固载到目标细胞的表面,经硝酸溶液处理之后释放出量子点中的Cd2+和pb2+,采用阳极溶出伏安法测定离子并得到相应的信号,信号处理之后便得到了关于目标肿瘤细胞及其标志物的信息。实验检测的灵敏度比较高,要归功于实验中所制备的纳米探针具有很好地放大效应。
Background
Tumors are divided into benign and malignant types. Generally speaking, the latter is what we often call cancer. In recent years, along with the increase of environmental pollution and food safetythe incidence of and mortality induced by cancer are increasing annually. Cancer has a reputation as a deadly disease.Paraneoplastic syndrome generally refers to the abnormal immune responses caused by unusual product of tumor or some relevant clinical manifestations in digestive, endocrine nervous, hematopoiesis, bone, kidney and skin system with unknown cause. These phenomenaare generally not directly caused by the primary tumor but through indirect routes so these syndrome are called paraneoplastic syndrome.
Paraneoplastic neurologic syndromes,(NPS) occurs in some patients with malignant tumor, and can affect any part of the central and peripheral nervous system without tumor metastasis.. PNS is not caused by the tumor direct invasion or its metastasis, or by metabolic disruptions, nutrition, infection or anti-tumor therapy, It is often marked by functional disorder of muscle, peripheral nerve, even spinal cord or brain. PNS is rare, affecting less than1%patients with cancer. However, PNS has a wide range of lesions and diverse clinical manifestations. In most NPS patients, the neurological disorder may develop before or after the cancer becomes clinically overt. According to statistics, about80%of patients, the paraneoplastic neurological syndrome (PNS), antedates the diagnosis of cancer. This provides an opportunity for us to discover the existence of malignant tumor. For those cancers with a serious threat to human life and health early discovery, diagnosis and treatment can significantly improve the survival of patients.
Compared to the symptoms of general nervous system tumor the paraneoplastic neurological syndrome (PNS) incidence is relatively low. Compared with complications in nervous system induced by other organ tumor, paraneoplastic syndrome is rare. But because of various factors, it is very important to early discover and treat paraneoplastic neurological syndrome (PNS) clinically. First, PNS may cause more serious damage and function defect, and the disorder may last longer; Secondly, for regular system patients with nervous system disease who show the phenomena of PNS,, it is often characteristic of that the carcinoma in situ is not yet diagnosed.. Therefore, as far as the neurologist is concerned, this is a kind of effective way to diagnose the disease in an early stage. The doctor needs to have a high ability to recognize and diagnose cancer. Again, if PNS could be found and treated in an early stage, the possibility to cure cancer could be greatly improved, and the function of the nervous system could be restored gradually.
Currently there are no particularly effective therapeutic agents to cure PNS. The current therapy includes vitamins, corticosteroids, plasma exchange and immunosuppressant treatmentbut their effects remain to be proved. Clinical studies demonstrated that the neurological paraneoplastic syndrome were relieved and improved after the primary tumor was effectively cured
The main innovation points of this research:
1. A novel quantum dot nanoparticles was designed in this study. Our study used aptamer DNA as stabilizer in the process of preparation of water-soluble nanoparticles and as the compound that participate in the reaction. This protocol simplifies the cumbersome steps required in the general protocol which needs to separately conjugate aptamer DNA to the surface of nanoparticles.2. The surface of a single quantum dot nanoparticle can have multiple DNA aptamers while in the surface of a single microspheres carrier can be combined with multiple quantum dot nanoparticles. so it has a function of cascade amplification, significantly improving the efficiency and sensitivity of detection.
PART I The detection of biomarkers in Neurological paraneoplastic syndrome
Objective:To investigate the early detection of the biomarkers in neurological paraneoplastic syndrome (paraneoplastic neurologic syndrome, PNS) and to study the early diagnosis and early preventive treatment.
Methods:
Information was obtained from our hospital between Sep2010and May2013. The32outpatients include12cases with PNS and20cases without PNS. Twelve PNS patients include7male and5female,,35to72years old with mean age49.3±11.7. old., The time of disease course was from3months to5years. The pathological types are small cell lung cancer SCLC) with5cases, breast cancer with3cases, ovarian cancer with2cases, liver cancer with1, pheochromocytoma with1. The20cases without NPS include12male and8female, aged38to75with mean age50.5±9.1, The time of disease course was from five months-4.5years. There are5cases of gastric carcinoma,5cases of systemic lupus erythematosus (sle),3of rheumatoid arthritis,3cases of prostate cancer,2dermatomyositis cases and2cases of pulmonary tuberculosis. All cases are confirmed by imaging, hematology and pathological examination. Other randomly selected20healthy people who are served as control group include10male and10female, with age from26to65and mean age40.1±9.2.
Age, sex, the time of disease course and other laboratory tests (including the electromyography, eeg, etc.) and imaging characteristics (including CT, MRI, enhanced MRI, MRA, etc were analyzed and evaluated. All healthy people and patients were subjected to clinical examination and detection of serum antibodies such as anti-Hu, anti-yo anti-Ri by enzyme-linked immunosorbent assay. We evaluated the relevance between the concentration and titer of specific antibodies and their and the primary lesions of PNS.
Results:
(1) No difference regarding the regular information The number of casesage, gender, time of disease course between two groups showed no statistically significant difference by T-test.(P>0.05).
(2) Enzyme-linked immunosorbent assay results:
Enzyme-linked immunosorbent assay results show:The expression of antibodies such as anti-Hu, anti-yo anti-Ri in group PNS was increased significantly than in group non-PNS (P<0.01)
(3) anti-Hu antibody in the diagnosis of NPS: According to diagnostic criteria for NPS by Graus,9of12cases were diagnosed as PNS or tumor,2cases (see table1. NO.9and12) without the primary tumor and the other7cases with tumor. The detection of Hu antibody may predict75%of patients with PNS (9/12)(see table2).
(4) the relationship between neurons antibody and NPS clinical types: NPS with positive anti-Hu mainly manifested as tumor sensory neuron disease (paraneoplastic chipmaker neuronopathy, PSN) and deputy tumor encephalomyelitis (paraneoplastic encephalomyelitis, P EM), with a few paraneoplastic limbic encephalitis(PL E) and paraneoplastic cerebellar degeneration(PCD) paraneoplastic chipmaker-motor neuropathy,(PSMN), etc.; Anti-Yo is mainly related to PCD yukio okamoto, antibody and Anti-Ri antibody is associated with paraneoplastic opsoclonus myoclonus(POM).
Conclusion:
1. The main biological markers of neurological paraneoplastic syndrome are anti Hu antibody, anti-yukio okamoto and anti-Ri antibody.
2. Enzyme-linked immunosorbent assay results show:The expression of antibodies such as anti-Hu, anti-yo anti-Ri in group PNS was increased significantly than in group non-PNS (P<0.01)
3. Detection of anti-Ri antibody,anti-Hu antibody and anti-Yo antibody in the sera of patients, can improve early detection of tumor cells. It can also provide early treatment to diagnostic basis for clinical.
PART Ⅱ
Electrochemical biosensor detection based on quantum dots and markers of tumor cells
Objective:
By improving the method of preparation of the DNA aptamer modified quantum dot nanoparticles and the probe after using anodic stripping voltammetry to detect specific ion in order to achieve the target tumor cells and tumor cell surface markers detection.
Methods:
PbS quantum dots and the preparation of CdS quantum dots is in reference to the related literature, the synthetic process for the improvement of synthetic method. First will lead nitrate solid powder and solid powder mixed with a certain concentration of cadmium chloride solution, And then in turn add thioglycolic acid and connection DNA, Half an hour after adding suitable amount of Na2S solution of24h corresponding quantum dot nanoparticles can be got. The whole experiment process was conducted under the condition of nitrogen protection of, the product obtained4°C saved for later use.
Preparation of divided into magnetic probes and non magnetic probe. Magnetic microspheres and the non magnetic polystyrene microspheres are cleaned with PBS buffer solution before use. Then prepared beforehand (MUC1) optimal solution and the preparation of the CdS quantum dots in magnetic beads system, Will its ehrs-3aptamer solution and preparation in advance of the PbS system of quantum dots in polystyrene microspheres, Thermostatic incubation can get corresponding nano probe.
Eventually adopted by the tumor cell electrochemical detection method for the anodic stripping voltammetry (DPV). With nitric acid solution will be immobilized on the surface of the tumor cells, probe from down and release the Cd2+and Pb2+. Will be prepared of glassy carbon electrode for the electrolytic cell, and then remove the electrode and in the samples are ready for testing system, begin to differential pulse voltammetric scans, set the leave time to15seconds,50mV pulse amplitude, pulse width50ms, potential scan range is0.30to0.80V (vs. Ag/AgCl). The potential of0.65V and0.45V anodic stripping peak current of Cd2+and Pb2+respectively the anodic dissolution of the signal.
Results:
Through continued optimization of experimental conditions eventually lead sulfide quantum dots and cadmium sulfide quantum dot the best preparation conditions of these two kinds of quantum dots. The optimum condition for synthesis of CdS quantum dots for10ml concentration of1.0×10-3M cadmium chloride solution, concentration of10ml of1.34x10-3M sodium sulphide solution, stabilizer for1mu L thioglycolic acid (C2H4O2S) and300mu L1.0×10-6M connection DNA; The optimum condition for synthesis of PbS quantum dots for10ml concentration for2×10-4M lead nitrate solution, concentration of4ml of6.7×10-4M sodium sulphide solution, stabilizer for1mu L thioglycolic acid (C2H4O2S) and300mu L1.0×10-6M connection DNA.
Through continued optimization of experimental conditions the resulting in the best test condition is:in the process of electrochemical detection of DNA aptamer optimal concentration of4.0×10-7M; The optimal pH value of4.7; Probe preparation for30h incubation time.
Finally detection of tumor cells in cell concentration1.0x102cells mL-2-1.0×106cells mL-1range, electrochemical detection signal to numerical proportional relationship with cell concentration, The calibration curves for the I (mu)=14.0024+9.7932lgC (cell mL-1), in which I deducted for blank current after the current intensity, C is the concentration of the cells in the cell SAP, linear correlation coefficient is0.995, n=9. Further calculated the detection limit of31.7cells mL-1; A single target cells (MUC1) on the surface of the target protein is about6.41x104, the number of single target cells on the surface of its ehrs-3the number of target protein is about3.47x104. At the same time, the test method has been verified by the experiment in the mixed samples are even more complex environment can still achieve good detection result.
Conclusion:
This study proposes a new method of detection of tumor cells and tumor markers. Through the setting of biological sensors, combining quantum dots and biological sensors, and then fixed load to the two magnetic beads and polystyrene microspheres carrier, they produced a type of two quantum dots based on CdS and PbS new electrochemical biosensors. Combining biological sensor and DNA aptamer again, it gives the biosensor specificity of certain goals. In this experiment, we connected on the biosensor (MUC1) and its ehrs-3of these two kinds of suitable body and used to analyze the detection of human breast cancer cells (MCF-7).
Using connection DNA as a synthesis of quantum dots stabilizer is one of the features of the experiment, so it makes quantum dots solid load to magnetic beads or polystyrene microspheres were possible. Then two kinds of DNA aptamer respectively connected to the quantum dots and modified magnetic beads made of polystyrene microsphere surface nano probe. Then through suitable body on the specificity of the target protein recognition probes immobilized onto the surface of the target cells, with aqueous nitric acid after releasing Cd2+and Pb2+in quantum dots, the anodic stripping voltammetry was developed for the determination of ion and get the corresponding signal, the signal processing after they got the information about target tumour cells and their markers. Experimental detection sensitivity is higher, thanks to the experiment of preparation of nano probe has amplification effect very well.
神经系统副肿瘤综合征(paraneoplastic neurologic syndrome, NPS)是发生在某些恶性肿瘤患者体内,在肿瘤没有发生转移的情况下,而引起自身其它部分或者器官的神经系统病变。它既非肿瘤的直接浸润或者转移,也非代谢、营养、感染以及抗肿瘤治疗引起。其表现常为肌肉,周围神经,甚至脊髓或者脑的功能性障碍。神经系统综合征(PNS)在恶性肿瘤患者中发病率一般<1%,但是病变范围较广,临床上表现多样。神经系统副肿瘤综合征(PNS)明显的神经系统表现可以发生在患者恶性肿瘤发生之前,同时或者之后。但通常来讲,神经系统副肿瘤综合征(PNS)一般发生在患者肿瘤发生之前较多一些。据统计约有80%左右的神经系统副肿瘤综合征(PNS)患者在肿瘤发生之前就有了明显的神经系统病变表现。这就为我们及早的发现恶性肿瘤的存在提供了一个契机。对于严重威胁人类生命健康的癌症,如果能及早的发现以及诊断治疗,那么就可以显著地提高患者的生存期。
相对于一般的神经系统肿瘤症状来说,虽然神经系统副肿瘤综合症(PNS)的发病率较低,与其他系统肿瘤的神经系统非转移性并发症相比,副肿瘤综合征也很少见,但是因为种种因素,神经系统副肿瘤综合征(PNS)就临床上而言,及早的发现和治疗还是非常重要的。首先,神经系统副肿瘤综合征的出现会对神经系统的功能的缺损造成更加严重的伤害,并且病症的持续时间更加长久；其次,对于一般的神经系统病症患者出现的副肿瘤综合征现象,其往往是一种还没能够诊断出来的原位癌的特征,因此,这对于神经科医生而言,这是对患者能够及早的诊断病症的一种有效途径,同时,这也需要医生具有较高的敏锐的识别能力和诊断能力；再次,如果能够早期发现诊断并且治疗副肿瘤综合征,可以使肿瘤患者治愈的可能性大大提高,并且还可以逐步恢复神经系统功能。
目前来讲,神经系统副肿瘤综合征还没有特别有效的治疗方法。现在通常用维生素类的药物,皮质类固醇,血浆置换以及免疫抑制剂等方法进行治疗,但是疗效均未得到证实。据一些临床治疗结果表明,部分患者在对原发肿瘤进行有效治疗后,其神经系统副肿瘤综合征的症状得到明显的缓解和好转。
本研究的主要创新点：
1.本研究设计了一种新颖的量子点纳米粒子。直接用适体DNA作为水溶性纳米粒子反应制备过程中的稳定剂和参与反应的化合物,简化了一般实验需要单独将适体DNA修饰到纳米粒子表面上的繁琐步骤。
2.由于在单个量子点纳米粒子表面可以同时具有多个适体DNA,且在单个微球载体表面可以同时结合多个量子点纳米粒子,所以这具有一种放大作用,显著地提高了检测的效率和灵敏度。
第一部分：神经系统副肿瘤综合征的生物学标记物检测
目的：研究神经系统副肿瘤综合征(paraneoplastic neurologic syndrome, PNS)早期生物学标记物的检测,探讨其早期的诊断和早期的预防性治疗。
方法：
资料来源于2010年09月至2013年05月期间在我院病房及门诊就诊的患者32例,其中PNS患者12例,无PNS的其它疾患者20例。12例的PNS患者中男7例,女5例,年龄在35-72岁,平均年龄49.3±11.7岁,发病病程在3月-5年,其中小细胞肺癌(small-cell lung cancer,SCLC)5例,乳腺癌3例,卵巢癌2例,肝癌1例,嗜铬细胞瘤1例；20例的无NPS的其它疾患者中男12例,女8例,年龄在38-75岁,平均年龄50.5±9.1岁,发病病程在5月-4.5年,其中胃癌5例,系统性红斑狼疮5例,类风湿性关节炎3例,前列腺癌3例,皮肌炎2例,肺结核2例。所有诊断均经影像学、血液学相关检测及病理检查予以确认。另选取20例的同期健康体检者,其中男10例,女10例,年龄在26-65岁,平均年龄40.1±9.2岁。
对所有患者的年龄、性别、疾病病程、其它实验室检查(包括肌电图、脑电图等)、影像学特点(包括CT、M R I、增强M R I、MRA等)等因素进行分析评价。将所有的入选对象进行相关的临床检查及酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)定量检测患者血清的抗-Hu抗体、抗-Yo抗体、抗-Ri抗体。并将特异性抗体的检测的高低与神经系统副肿瘤综合征的原发病灶及临床表现进行比较及评价。
结果：
(1)两组患者的一般资料比较结果：
两组患者的病例数、年龄、性别、疾病病程等一般指标经t检验表明,两组间差异无统计学意义(P>0.05)。
(2) ELISA定量检测实验结果：
本研究ELISA结果显示PNS组抗-Hu抗体、抗-Ri抗体、抗-Yo抗体表达较非PNS组显著升高,统计有显著差异性(P<0.01)。
(3)特异性抗-Hu抗体在NPS诊断中的阳性预测值：
根据Graus等的NPS诊断标准,12例中9例确诊PNS或发现相关肿瘤,其中2例(见表1中的病例9和病例12)未发现原发肿瘤,另7例发现肿瘤。抗Hu抗体在NPS诊断中的阳性预测值为75%(9/12)。
(4)神经元抗体与NPS临床类型之间的关系：
抗-Hu抗体阳性的NPS患者主要表现为副肿瘤性感觉性神经元病(paraneoplastic sensory neuronopathy, PSN)和副肿瘤性脑脊髓炎(paraneoplastic encephalomyelitis, P EM),少数可表现为副肿瘤性边缘叶脑炎(paraneoplastic limbic encephalitis, PL E)、副肿瘤性小脑变性(paraneoplastic cerebellar degeneration, PCD)和副肿瘤性感觉运动性神经病(paraneoplastic sensory-motor neuropathy, PSMN)等；抗-Yo抗体主要导致PCD；抗-Ri抗体主要与副肿瘤性眼阵挛-肌阵挛(paraneoplastic opsoclonus-myoclonus, POM)有关。
结论：
1.神经系统副肿瘤综合征的主要生物学标记物为抗-Hu抗体、抗-Yo抗体、抗-Ri抗体三种抗体。
2. ELISA结果显示PNS组抗-Hu抗体、抗-Ri抗体、抗-Yo抗体表达较非PNS组显著升高,统计有显著差异性(P<0.01)。
3.检测患者血清的抗-Hu抗体、抗-Yo抗体、抗-Ri抗体,以此来提高肿瘤细胞早期检测,为临床的早期治疗提供诊断依据。
第二部分：基于量子点的电化学生物传感器检测肿瘤细胞及其标志物
目的：
通过改进的方法制备经适体DNA修饰过量子点纳米粒子和探针之后,采用阳极溶出伏安法对特定离子的检测以达到对目标肿瘤细胞以及肿瘤细胞表面的的标志物检测。进而为医学上成功检测肿瘤细胞提供一种可行的有效手段
方法：
硫化铅量子点和硫化镉量子点的制备是在参照了相关文献之后,对其合成工艺做了改进而得出的合成方法。首先将硝酸铅固体粉末和氯化镉固体粉末配制成一定浓度的溶液,然后依次加入巯基乙酸和连接DNA反应,半小时后再加入适量Na2S溶液反应24h便可得到相应的量子点纳米粒子。整个实验过程是在通氮气保护的条件下进行的,所得产物4℃保存备用。
探针的制备分为磁性探针和非磁性探针两种。磁性微球与非磁性聚苯乙烯微球在使用之前均用PBS缓冲溶液清洗。然后将事先准备好的MUC1适体溶液和事先制备的CdS量子点于磁珠体系当中,将HER-3适体溶液和事先制备的PbS量子点于聚苯乙烯微球体系当中,恒温孵育便可得到相应的纳米探针。
最终的电化学检测肿瘤细胞所采用的方法为阳极溶出伏安法(DPV)。用硝酸溶液将固载在肿瘤细胞表面的探针脱离下来并释放出Cd2+和pb2+。将事先准备好的玻碳电极至于电解池当中,然后取出电极并置于已经准备好的待测样品体系当中,开始进行差分脉冲伏安扫描,设置静置时间为15秒,脉冲增幅50mV,脉冲宽度50ms,电位扫描范围为-0.30to-0.80V(vs. Ag/AgCl)。在电位-0.65V和-0.45V所出现的阳极溶出峰值电流分别为Cd2+和pb2+的阳极溶出信号。
结果：
通过对实验条件的不断优化最终得到硫化铅量子点和硫化镉量子点这两种量子点的最佳制备条件。合成CdS量子点的最优条件为10mL浓度为1.0×10-3M的氯化镉溶液,10mL浓度为1.34×10-3硫化钠溶液,稳定剂为1μL基乙酸(C2H4O2S)和300μL1.0×10-6M连接DNA；合成PbS量子点的最优条件为10mL浓度为2×10-4M的硝酸铅溶液,4mL浓度为6.7×10-4M硫化钠溶液,稳定剂为1μL巯基乙酸(C2H4O2S)和300μL1.0×10-6M连接DNA。
通过对实验条件的不断优化最终得到在电化学检测过程中的最佳检测条件为：适体DNA的最佳浓度为4.0×10-7M；最佳pH值为4.7；探针制备孵育时间为30h。
最终对肿瘤细胞的检测,在细胞浓度1.0×102cells mL-1—1.0×106cells mL-1的变化范围内,电化学检测信号与细胞浓度的对数值成正比例关系,其校正曲线为I(μA)=-14.0024+9.7932lgC(cell mL-1),其中I为扣除空白电流之后的电流强度,C为细胞液中细胞的浓度,线性相关系数为0.995,n=9。进一步计算得到其检测限为31.7cells mL-1；单个靶细胞表面上的MUC1目标蛋白的数量约为6.41×104个,单个靶细胞表面上的HER-3目标蛋白的数量约为3.47x104个。同时,这种检测方法经实验证实在混合样品甚至更加复杂的环境中依然可以达到很好的检测效果。
结论：
本实验提出了一种全新的检测肿瘤细胞以及肿瘤标记物的方法。通过对生物传感器的设置,将量子点与生物传感器相结合,最后再固载至磁珠和聚苯乙烯微球这两种载体上,制成了一种基于CdS和PbS两种量子点的新型的电化学生物传感器。再将生物传感器与适体DNA相结合,这就赋予了生物传感器对特定目标的专一性。本实验中,我们在生物传感器上连接了MUC1和HER-3这两种适体,用于分析检测人类乳腺癌细胞(MCF-7)。
利用连接DNA作为合成量子点的稳定剂是本实验的一大特色,因为有它的存在才使得将量子点固载至磁珠或者聚苯乙烯微球上成为可能。然后将两种适体DNA分别连接到经量子点修饰过的磁珠和聚苯乙烯微球表面制成纳米探针。然后通过适体对目标蛋白的特异性识别是的探针固载到目标细胞的表面,经硝酸溶液处理之后释放出量子点中的Cd2+和pb2+,采用阳极溶出伏安法测定离子并得到相应的信号,信号处理之后便得到了关于目标肿瘤细胞及其标志物的信息。实验检测的灵敏度比较高,要归功于实验中所制备的纳米探针具有很好地放大效应。
Background
Tumors are divided into benign and malignant types. Generally speaking, the latter is what we often call cancer. In recent years, along with the increase of environmental pollution and food safetythe incidence of and mortality induced by cancer are increasing annually. Cancer has a reputation as a deadly disease.Paraneoplastic syndrome generally refers to the abnormal immune responses caused by unusual product of tumor or some relevant clinical manifestations in digestive, endocrine nervous, hematopoiesis, bone, kidney and skin system with unknown cause. These phenomenaare generally not directly caused by the primary tumor but through indirect routes so these syndrome are called paraneoplastic syndrome.
Paraneoplastic neurologic syndromes,(NPS) occurs in some patients with malignant tumor, and can affect any part of the central and peripheral nervous system without tumor metastasis.. PNS is not caused by the tumor direct invasion or its metastasis, or by metabolic disruptions, nutrition, infection or anti-tumor therapy, It is often marked by functional disorder of muscle, peripheral nerve, even spinal cord or brain. PNS is rare, affecting less than1%patients with cancer. However, PNS has a wide range of lesions and diverse clinical manifestations. In most NPS patients, the neurological disorder may develop before or after the cancer becomes clinically overt. According to statistics, about80%of patients, the paraneoplastic neurological syndrome (PNS), antedates the diagnosis of cancer. This provides an opportunity for us to discover the existence of malignant tumor. For those cancers with a serious threat to human life and health early discovery, diagnosis and treatment can significantly improve the survival of patients.
Compared to the symptoms of general nervous system tumor the paraneoplastic neurological syndrome (PNS) incidence is relatively low. Compared with complications in nervous system induced by other organ tumor, paraneoplastic syndrome is rare. But because of various factors, it is very important to early discover and treat paraneoplastic neurological syndrome (PNS) clinically. First, PNS may cause more serious damage and function defect, and the disorder may last longer; Secondly, for regular system patients with nervous system disease who show the phenomena of PNS,, it is often characteristic of that the carcinoma in situ is not yet diagnosed.. Therefore, as far as the neurologist is concerned, this is a kind of effective way to diagnose the disease in an early stage. The doctor needs to have a high ability to recognize and diagnose cancer. Again, if PNS could be found and treated in an early stage, the possibility to cure cancer could be greatly improved, and the function of the nervous system could be restored gradually.
Currently there are no particularly effective therapeutic agents to cure PNS. The current therapy includes vitamins, corticosteroids, plasma exchange and immunosuppressant treatmentbut their effects remain to be proved. Clinical studies demonstrated that the neurological paraneoplastic syndrome were relieved and improved after the primary tumor was effectively cured
The main innovation points of this research:
1. A novel quantum dot nanoparticles was designed in this study. Our study used aptamer DNA as stabilizer in the process of preparation of water-soluble nanoparticles and as the compound that participate in the reaction. This protocol simplifies the cumbersome steps required in the general protocol which needs to separately conjugate aptamer DNA to the surface of nanoparticles.2. The surface of a single quantum dot nanoparticle can have multiple DNA aptamers while in the surface of a single microspheres carrier can be combined with multiple quantum dot nanoparticles. so it has a function of cascade amplification, significantly improving the efficiency and sensitivity of detection.
PART I The detection of biomarkers in Neurological paraneoplastic syndrome
Objective:To investigate the early detection of the biomarkers in neurological paraneoplastic syndrome (paraneoplastic neurologic syndrome, PNS) and to study the early diagnosis and early preventive treatment.
Methods:
Information was obtained from our hospital between Sep2010and May2013. The32outpatients include12cases with PNS and20cases without PNS. Twelve PNS patients include7male and5female,,35to72years old with mean age49.3±11.7. old., The time of disease course was from3months to5years. The pathological types are small cell lung cancer SCLC) with5cases, breast cancer with3cases, ovarian cancer with2cases, liver cancer with1, pheochromocytoma with1. The20cases without NPS include12male and8female, aged38to75with mean age50.5±9.1, The time of disease course was from five months-4.5years. There are5cases of gastric carcinoma,5cases of systemic lupus erythematosus (sle),3of rheumatoid arthritis,3cases of prostate cancer,2dermatomyositis cases and2cases of pulmonary tuberculosis. All cases are confirmed by imaging, hematology and pathological examination. Other randomly selected20healthy people who are served as control group include10male and10female, with age from26to65and mean age40.1±9.2.
Age, sex, the time of disease course and other laboratory tests (including the electromyography, eeg, etc.) and imaging characteristics (including CT, MRI, enhanced MRI, MRA, etc were analyzed and evaluated. All healthy people and patients were subjected to clinical examination and detection of serum antibodies such as anti-Hu, anti-yo anti-Ri by enzyme-linked immunosorbent assay. We evaluated the relevance between the concentration and titer of specific antibodies and their and the primary lesions of PNS.
Results:
(1) No difference regarding the regular information The number of casesage, gender, time of disease course between two groups showed no statistically significant difference by T-test.(P>0.05).
(2) Enzyme-linked immunosorbent assay results:
Enzyme-linked immunosorbent assay results show:The expression of antibodies such as anti-Hu, anti-yo anti-Ri in group PNS was increased significantly than in group non-PNS (P<0.01)
(3) anti-Hu antibody in the diagnosis of NPS: According to diagnostic criteria for NPS by Graus,9of12cases were diagnosed as PNS or tumor,2cases (see table1. NO.9and12) without the primary tumor and the other7cases with tumor. The detection of Hu antibody may predict75%of patients with PNS (9/12)(see table2).
(4) the relationship between neurons antibody and NPS clinical types: NPS with positive anti-Hu mainly manifested as tumor sensory neuron disease (paraneoplastic chipmaker neuronopathy, PSN) and deputy tumor encephalomyelitis (paraneoplastic encephalomyelitis, P EM), with a few paraneoplastic limbic encephalitis(PL E) and paraneoplastic cerebellar degeneration(PCD) paraneoplastic chipmaker-motor neuropathy,(PSMN), etc.; Anti-Yo is mainly related to PCD yukio okamoto, antibody and Anti-Ri antibody is associated with paraneoplastic opsoclonus myoclonus(POM).
Conclusion:
1. The main biological markers of neurological paraneoplastic syndrome are anti Hu antibody, anti-yukio okamoto and anti-Ri antibody.
2. Enzyme-linked immunosorbent assay results show:The expression of antibodies such as anti-Hu, anti-yo anti-Ri in group PNS was increased significantly than in group non-PNS (P<0.01)
3. Detection of anti-Ri antibody,anti-Hu antibody and anti-Yo antibody in the sera of patients, can improve early detection of tumor cells. It can also provide early treatment to diagnostic basis for clinical.
PART Ⅱ
Electrochemical biosensor detection based on quantum dots and markers of tumor cells
Objective:
By improving the method of preparation of the DNA aptamer modified quantum dot nanoparticles and the probe after using anodic stripping voltammetry to detect specific ion in order to achieve the target tumor cells and tumor cell surface markers detection.
Methods:
PbS quantum dots and the preparation of CdS quantum dots is in reference to the related literature, the synthetic process for the improvement of synthetic method. First will lead nitrate solid powder and solid powder mixed with a certain concentration of cadmium chloride solution, And then in turn add thioglycolic acid and connection DNA, Half an hour after adding suitable amount of Na2S solution of24h corresponding quantum dot nanoparticles can be got. The whole experiment process was conducted under the condition of nitrogen protection of, the product obtained4°C saved for later use.
Preparation of divided into magnetic probes and non magnetic probe. Magnetic microspheres and the non magnetic polystyrene microspheres are cleaned with PBS buffer solution before use. Then prepared beforehand (MUC1) optimal solution and the preparation of the CdS quantum dots in magnetic beads system, Will its ehrs-3aptamer solution and preparation in advance of the PbS system of quantum dots in polystyrene microspheres, Thermostatic incubation can get corresponding nano probe.
Eventually adopted by the tumor cell electrochemical detection method for the anodic stripping voltammetry (DPV). With nitric acid solution will be immobilized on the surface of the tumor cells, probe from down and release the Cd2+and Pb2+. Will be prepared of glassy carbon electrode for the electrolytic cell, and then remove the electrode and in the samples are ready for testing system, begin to differential pulse voltammetric scans, set the leave time to15seconds,50mV pulse amplitude, pulse width50ms, potential scan range is0.30to0.80V (vs. Ag/AgCl). The potential of0.65V and0.45V anodic stripping peak current of Cd2+and Pb2+respectively the anodic dissolution of the signal.
Results:
Through continued optimization of experimental conditions eventually lead sulfide quantum dots and cadmium sulfide quantum dot the best preparation conditions of these two kinds of quantum dots. The optimum condition for synthesis of CdS quantum dots for10ml concentration of1.0×10-3M cadmium chloride solution, concentration of10ml of1.34x10-3M sodium sulphide solution, stabilizer for1mu L thioglycolic acid (C2H4O2S) and300mu L1.0×10-6M connection DNA; The optimum condition for synthesis of PbS quantum dots for10ml concentration for2×10-4M lead nitrate solution, concentration of4ml of6.7×10-4M sodium sulphide solution, stabilizer for1mu L thioglycolic acid (C2H4O2S) and300mu L1.0×10-6M connection DNA.
Through continued optimization of experimental conditions the resulting in the best test condition is:in the process of electrochemical detection of DNA aptamer optimal concentration of4.0×10-7M; The optimal pH value of4.7; Probe preparation for30h incubation time.
Finally detection of tumor cells in cell concentration1.0x102cells mL-2-1.0×106cells mL-1range, electrochemical detection signal to numerical proportional relationship with cell concentration, The calibration curves for the I (mu)=14.0024+9.7932lgC (cell mL-1), in which I deducted for blank current after the current intensity, C is the concentration of the cells in the cell SAP, linear correlation coefficient is0.995, n=9. Further calculated the detection limit of31.7cells mL-1; A single target cells (MUC1) on the surface of the target protein is about6.41x104, the number of single target cells on the surface of its ehrs-3the number of target protein is about3.47x104. At the same time, the test method has been verified by the experiment in the mixed samples are even more complex environment can still achieve good detection result.
Conclusion:
This study proposes a new method of detection of tumor cells and tumor markers. Through the setting of biological sensors, combining quantum dots and biological sensors, and then fixed load to the two magnetic beads and polystyrene microspheres carrier, they produced a type of two quantum dots based on CdS and PbS new electrochemical biosensors. Combining biological sensor and DNA aptamer again, it gives the biosensor specificity of certain goals. In this experiment, we connected on the biosensor (MUC1) and its ehrs-3of these two kinds of suitable body and used to analyze the detection of human breast cancer cells (MCF-7).
Using connection DNA as a synthesis of quantum dots stabilizer is one of the features of the experiment, so it makes quantum dots solid load to magnetic beads or polystyrene microspheres were possible. Then two kinds of DNA aptamer respectively connected to the quantum dots and modified magnetic beads made of polystyrene microsphere surface nano probe. Then through suitable body on the specificity of the target protein recognition probes immobilized onto the surface of the target cells, with aqueous nitric acid after releasing Cd2+and Pb2+in quantum dots, the anodic stripping voltammetry was developed for the determination of ion and get the corresponding signal, the signal processing after they got the information about target tumour cells and their markers. Experimental detection sensitivity is higher, thanks to the experiment of preparation of nano probe has amplification effect very well.
引文
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[4]Candler PM, Hart PE, Barnett M et al A follow up study of patients with paraneoplastic neurological disease in the United Kingdom 1JNeurolNeurosurg Psychiatry,2004; 75(10):1411-1415
[5]王彬,查建平.副肿瘤性小脑病变与抗神经元抗体关系[J].神经疾病与精神卫生,2010,10(1)：44-46.
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[7]Graus F, Delattre JY, Antoine JC et all Recommended diagnostic ccriteria for paraneoplastic neurological syndromes 1J Neurol Neurosurg Psychiatry,2004; 75 (8):1135-1140.
[8]Toothaker TB, RubinM.Paraneoplastic neurological syndromes:a review. Neurologist,2009,15:21-33.
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