外泌体基因检测在结直肠癌治疗中的应用

发布时间：2018-05-31 00:00

本文选题：外泌体 + 稳定性　；参考：《中国人民解放军军事医学科学院》2017年博士论文

【摘要】：肿瘤的个体化治疗是以患者的药物基因组学为基础,制定针对每个患者自身合理的治疗方案,以期最大限度的获得临床疗效并降低毒副作用治疗模式。个体化治疗的前提条件是要进行肿瘤组织的病理学基因检测,但是由于肿瘤早期不易发现、取材困难以及肿瘤异质性等原因,患者的诊断及后续治疗受到了诸多方面的限制。因此,寻找一种简便易得同时与肿瘤组织基因变化相一致的替代物成为个体化治疗的重要任务。外泌体(exosomes)是一类起源于细胞内吞体系统的纳米级小囊泡,直径在30-150nm之间,在电镜下呈现球形或杯状的形态,拥有稳定的双层膜结构,通过胞吐作用排出细胞外,并随之进入血流或其他体液系统中。人体内几乎所有细胞都能分泌产生外泌体。外泌体几乎分布于人体内所有的体液系统,是细胞间沟通联系的桥梁。研究发现,肿瘤患者体内的外泌体数量远高于健康人,更为重要的是外泌体内含有双链DNA,并且这些DNA所携带的基因信息在某种程度上与肿瘤细胞DNA具有一致性。本研究旨在探索结直肠癌患者血清外泌体中DNA的突变情况与肿瘤组织基因突变之间的相互关系,进而论证外泌体作为一种新型基因检测手段指导个体化治疗的可行性。外泌体如果想成为基因检测的理想材料,除了取材方便、含量丰富之外必须具备相当程度的稳定性,这是一切后续工作的前提和基础。所以在本文第一章中我们着重讨论外泌体的稳定性。我们将新鲜采集的血清样本分装后分别储存在4°C(24h,72h,168h);室温(6h,12h,24h,48h);-80°C反复冻融(1次,3次,5次)的条件下。用新鲜分离的血清作为对照,分别提取上述各个条件下的血清外泌体,同时保留去除外泌体之后的剩余血清。随后提取外泌体及剩余血清中的蛋白质和DNA,通过对外泌体特异性标志物TSG101和CD63蛋白的Western blot验证和DNA的PCR扩增及Sanger测序等方法分别对外泌体及外泌体DNA的稳定性进行了验证。结果发现,外泌体DNA是血清DNA的主要来源,并且无论外界环境如何变化,外泌体都能处于一个相对稳定的状态。这种稳定状态的持续时间随储存环境的变化而改变:从4°C储存的血清中提取得到的外泌体及其DNA最为稳定,这种稳定性可以持续至少1周的时间;在室温下外泌体至少可以稳定1天的时间;反复冻融对外泌体的破坏最为严重,冻融3次以后血清中的外泌体及其DNA被大量破坏。但是,即便是在室温放置48h乃至冻融5次的情况下,外泌体DNA依然能进行PCR扩增并检测出与新鲜血清外泌体一致的基因突变。以上结果不仅充分说明了外泌体及外泌体DNA具有极强的稳定性,而且提供了这种稳定性存在的持续时间,为外泌体作为一种新型的肿瘤基因检测手段提供了可靠的保证。为了研究外泌体内的基因状态是否与肿瘤组织基因检测的结果相一致,在本文第二章的实验中,我们随机收集了96例经病理组织学基因检测明确KRAS基因状态的结直肠癌患者的血清,分别进行了血清外泌体和血细胞DNA的提取。随后选取了与结直肠癌密切相关的6个基因8个基因位点(KRAS G12 G13,KRAS Q61,BRAF V600,NRAS Q61,PIK3CA E542 E545,PIK3CA H1047)分别进行了外泌体及血细胞DNA的PCR扩增、电泳验证以及扩增产物的高通量测序分析。与此同时,随机抽取了30例健康志愿者的血液样本作为对照,并对其进行外泌体及血细胞中KRAS G12 G13、KRAS Q61、BRAF V600基因位点扩增及二代测序。结果发现,外泌体DNA中KRAS基因野生型的患者,在肿瘤组织中同样为野生型;所有经组织学检测存在KRAS基因突变的患者在外泌体DNA中也都同样检测到了突变,但是对于组织学检测KRAS基因为野生型的患者来说,有一部分患者对应的外泌体基因检测却表现为突变型。上述这种情况在NRAS基因中也有所体现。对于PIK3CA基因来讲,三个位点在外泌体DNA中均存在较高比例的突变,特别是E545基因位点,外泌体中的突变率高达92.7%;另外该位点的血细胞DNA也有较高的突变率,其余7个基因位点很少能检测到血细胞DNA的突变。与PIK3CA基因不同,BRAF V600、KRAS Q61、NRAS Q61在外泌体中的突变率较低,基本与文献报道一致。而来自健康志愿者的血液样本中均没有检测到相关基因的突变。上述结果表明:对于结直肠癌患者来说,外泌体基因检测结果与肿瘤组织基因检测在某种程度上具有一致性,有可能作为一种潜在的生物标志物应用于临床检测。既然外泌体中存在基因突变且某些基因的突变比例相对较高,那么在结直肠癌患者中是否还存在其他的基因也有类似的高突变率;外泌体中的基因突变率是否会在患者临床治疗的过程中发生变化;是否这种变化可以提前预知患者对治疗的反应呢?带着上述疑问,我们收集了37例具有完整治疗过程的晚期结直肠癌患者在用药过程中的一系列血液样本,分别进行了血清外泌体DNA和血细胞DNA的提取。随后结合文献和COSMIC数据库中的大肠癌基因突变情况选取了与结直肠癌密切相关的24个基因30个基因位点分别进行了外泌体及血细胞DNA的PCR扩增、核酸电泳验证以及高通量测序分析。根据现阶段返回的测序结果有以下发现,首先,病理组织学基因检测明确KRAS基因为野生型的患者在治疗过程中血清外泌体DNA会发生KRAS基因的突变;其次,外泌体DNA中除了第二章中涉及到PIK3CA E545、PIK3CA H1047、KRAS G12 G13等基因位点同样具有高频突变以外,MAP2K1 K57基因也具有高频突变;并且,上述基因的突变率会随着患者的治疗过程不断发生改变,而这种变化的出现会早于影像学可见的改变,甚至早于患者治疗过程中肿瘤标志物的变化。因此,利用外泌体中基因的突变情况可能能够对患者的治疗效果进行预判,利用这种变化可以更好地指导临床治疗过程并及时调整用药方案,针对患者的全身肿瘤情况实施更为有效的个体化治疗。
[Abstract]:Individualized treatment of tumors is based on the pharmacogenomics of the patient, formulating a reasonable treatment plan for each patient to maximize the clinical efficacy and lower toxic and side effects therapy. The premise of individualized treatment is to carry out the pathological gene detection of tumor tissue, but because of the early tumor, It is easy to find that the diagnosis and subsequent treatment of the patients are limited by many reasons. Therefore, it is an important task to find a simple and convenient alternative that is consistent with the gene changes of the tumor tissue. The exocrine (exosomes) is a type of endocytic system that is derived from the endocytic system. A small nanoscale vesicle with a diameter between 30-150nm and a spherical or cup-shaped shape under electron microscopy. It has a stable double layer membrane structure that excrete into the blood flow or other body fluids through exocytosis. Almost all cells in the body can produce exocrine. The exocrine is almost distributed in all bodies in the body. The liquid system is a bridge of intercellular communication. It is found that the number of exocrine bodies in the tumor patients is much higher than that of the healthy people, and the more important is that the external secreting body contains double stranded DNA, and the gene information carried by these DNA is in some degree consistent with the tumor cell DNA. This study aims to explore the serum of patients with colorectal cancer. The relationship between the mutation of DNA and the mutation of the tumor tissue in the secreting body, and then demonstrate the feasibility of the exocrine as a new gene detection method to guide the individualized treatment. Exocrine, if it wants to be the ideal material for gene detection, must have a considerable degree of stability in addition to the convenience of the material. In the first chapter of this article, we focus on the stability of the exocrine. We store the fresh serum samples in 4 degrees C (24h, 72h, 168h); room temperature (6h, 12h, 24h, 48h); -80 degree C under repeated freezing and thawing (1 times, 3 times, 5 times). The fresh separated serum is used as a control, respectively. The serum Exocyst under the above conditions was taken and the remaining serum was retained after the exocrine removal. The protein and DNA in the exocrine and remaining serum were extracted, and the Western blot verification of the TSG101 and CD63 protein of the external secretory specific markers and the PCR amplification and Sanger sequencing of DNA were used to separate the secreting and exocrine D, respectively. The stability of NA is verified. It is found that exocrine DNA is the main source of DNA in serum, and the exocrine can be in a relatively stable state regardless of the environment. The duration of the stable state changes with the change of the storage environment: the exocrine extracted from the serum stored in 4 degree C and its DNA In order to stabilize the stability, the stability can last at least 1 weeks, and at least 1 days at room temperature can be stabilized at room temperature; repeated freezing and thawing the most serious damage to the external secreting body. After 3 times freeze thawing, the exocrine and its DNA in the serum are destroyed. However, the exocrine DNA remains even at room temperature for 48h and even for 5 times of freezing and thawing. PCR amplification and detection of gene mutations consistent with the fresh serum exocrine are performed. The above results not only fully demonstrate the strong stability of the exocrine and exocrine DNA, but also provide a lasting time for the existence of this stability, which provides a reliable guarantee for the exocrine as a new method for detecting tumor genes. In the second chapter of this study, 96 cases of colorectal cancer patients with clear KRAS gene status were collected and the serum exocyts and blood cells DNA were extracted. 6 gene loci closely related to colorectal cancer (KRAS G12 G13, KRAS Q61, BRAF V600, NRAS Q61, PIK3CA E542 E545) were amplified, electrophoretic verification and high throughput sequencing analysis of the amplified products. At the same time, blood samples from 30 healthy volunteers were randomly selected. In contrast, KRAS G12 G13, KRAS Q61, BRAF V600 loci amplification and two generation sequencing were carried out in the exocrine and blood cells. The results showed that the wild type of the KRAS gene in the exocrine DNA was also wild in the tumor tissue, and all the patients with the KRAS gene mutation in all histology tests were also detected in the exocrine DNA. The mutation was found, but for the KRAS gene of the wild type, some of the corresponding exocrine genes were found to be mutated. The above was also reflected in the NRAS gene. For the PIK3CA gene, the three loci had a high proportion of mutations in the exocrine DNA, especially the E545. In the gene locus, the mutation rate in the exocrine is up to 92.7%, and the blood cell DNA of the site also has a high mutation rate, and the other 7 loci rarely detect the mutation of the blood cell DNA. The mutation rate of BRAF V600, KRAS Q61 and NRAS Q61 in the exocrine is lower than that of the PIK3CA gene. The above results show that, for colorectal cancer patients, the exocrine gene detection results are in some degree consistent with the tumor tissue gene detection, and may be used as a potential biomarker for clinical detection. There is a gene process in the exocrine. If the mutation ratio of some genes is relatively high, there is a similar high mutation rate in the other genes in the patients with colorectal cancer; is the mutation rate in the exocrine in the process of the patient's clinical treatment; is this change early to predict the patient's response to the treatment? With the above suspicion We have collected a series of blood samples from 37 patients with advanced colorectal cancer in the process of complete treatment. The extraction of serum Exocyst DNA and blood cell DNA were carried out respectively. Then 24 genes closely related to colorectal cancer were selected in the literature and COSMIC database. The PCR amplification of exocrine and blood cells DNA, nucleic acid electrophoresis and high throughput sequencing analysis were carried out at the loci respectively. According to the results of the sequence returned at this stage, first, the histopathological gene detected that the KRAS gene of the wild type in the course of treatment could produce the KRAS gene in the serum exocrine DNA in the course of treatment. Secondly, in the exocrine DNA, in addition to the second chapters involving PIK3CA E545, PIK3CA H1047, KRAS G12 G13 and other gene loci, the MAP2K1 K57 gene also has a high frequency mutation, and the mutation rate of the above gene changes with the patient's treatment process, and this change will appear earlier than that of the imageology. The changes are even earlier than the changes in the tumor markers during the treatment of the patients. Therefore, the mutation of the genes in the exocrine may be able to prejudge the therapeutic effect of the patient, using this change to better guide the clinical treatment process and to adjust the drug prescription in time. Effective individualized treatment.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.34

【相似文献】