EML4-ALK融合基因的无创检测

发布时间：2018-04-01 08:19

本文选题：融合基因　切入点：EML4-ALK　出处：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：在对间变性大细胞淋巴瘤的研究中,研究者首次发现了间变性淋巴瘤激酶(Anaplastic lymphoma kinase,ALK)的存在。它是一种酪氨酸激酶受体(Receptor tyrosine kinase,RTKs)。由于染色体倒位的发生,ALK基因能够与棘皮动物微管相关类蛋白4(Echinoderm microtubule-associated protein-like 4,EML4)基因发生融合,形成EML4-ALK融合基因。ALK基因通过借助EML4的启动子,使ALK激酶活化,导致细胞发生癌变,促进癌症的发生和发展。目前研究发现,包括肺癌在内的多种实体瘤中均可检测到EML4-ALK融合基因的存在,其中,在非小细胞肺癌(non-small cell lung cancer,NSCLC)中的检出率最高,并且该类患者具有鲜明的临床特征,提示该靶点是肺腺癌特异性较高的分子标记物。目前,至少发现了14种EML4-ALK的变异体类型,所有这些融合基因的表达产物为一种嵌合型酪氨酸激酶,具有激酶活性,可激活ALK蛋白的胞内催化区域,激活相关信号通路,促进细胞增殖、存活、迁移,从而达到细胞癌变的效果。因此,快速、准确和及早地检测EML4-ALK融合基因对肺癌的早期检测、分子分型和靶向治疗至关重要。目前,所有EML4-ALK融合基因的检测方法都是针对手术或者活检取材后的肿瘤组织进行的,无法实现早期检测和实时监测,且由于肿瘤异质性问题,使得患者的检测结果可能出现假阴性。近年来,液态活检为肿瘤的检测提供新的思路,能够有效的避免组织活检的局限性。外泌体是液态活检主要检测的物质之一。它的形成过程:首先是由细胞通过内吞作用形成早期内体,而后早期内体经过内陷发展为多囊泡内体,当多囊泡内体转移至细胞膜附近时,其与细胞膜发生融合,从而导致原本存在于多囊泡内体中的囊泡释放到细胞外,通过这种方式分泌到胞外的囊泡称为外泌体。由于外泌体特殊的形成过程,使得其中含有能够反映分泌细胞生理状态的蛋白质、DNA和microRNA等信号分子,可能包含了细胞病变相关的信息。这一点使外泌体成为能够提供丰富的潜在生物标志物分子源的靶标。疾病的不同,病变细胞所分泌到胞外的外泌体中含有的特定成分不同。这使得从体液(包括血液、尿液、胸腹水等)中分离纯化外泌体,再对其组成及信号分子进行分析成为可能,有望应用于疾病诊断及监测,尤其在肿瘤标志物检测方面。因此,外泌体极有可能在一定条件下代替组织活检,成为一种新型无创的疾病检测靶标,并且可以获得疾病早期发展的相关信息。目前,关于外泌体中信号分子的报道有:来源于肿瘤细胞的蛋白质、双链DNA、microRNA等。但外泌体中是否含有融合基因的mRNA还尚未报道。目前具有该融合基因的细胞系有三种,分别为H3122、H2228以及DFCI032细胞。选取H3122细胞、H2228细胞以及不具有EML4-ALK融合基因的A549细胞作为阴性对照进行试验,对使用外泌体检测融合基因mRNA的可行性进行初步验证。首先在NCBI上查找EML4和ALK的基因序列,并针对这两种基因的几种不同融合形式分别设计引物。提取H3122、A549细胞的RNA进行逆转录PCR,测序结果证实H3122细胞发生融合的位点在EML4的第13外显子与ALK的第20外显子发生融合,即为V1融合形式。同时,提取这两种细胞的蛋白质进行Western Blot实验以检测ALK蛋白的表达情况。Western Blot实验结果显示,H3122细胞在120kD处有一条特异性条带,通过对EML4-ALK融合蛋白大小进行分析,发现其与V1形式的融合蛋白大小相符。将H3122、A549细胞饥饿处理24-48小时后,收集细胞培养上清,提取细胞培养上清中的外泌体。通过电镜形态学观察以及外泌体特异性表面膜蛋白的分析,证实外泌体提取成功。提取外泌体中的RNA,进行RT-PCR,在H3122细胞培养上清外泌体可以成功扩增到EML4-ALK融合基因,测序结果证实其融合形式也是V1,与H3122细胞中的融合形式是一致的。对H2228细胞使用相同的实验方法检测EML4-ALK融合基因及其融合形式,测序结果证实其融合形式为V3a(融合位点为EML4基因的第6外显子与ALK基因的第20外显子发生融合)和V3b(融合位点为EML4基因的第6外显子加上33bp内含子与ALK基因的第20外显子发生融合),在H2228细胞培养上清外泌体中也可以检测到EML4-ALK融合基因,其融合形式与细胞的融合形式是一致的。除了对肺癌细胞H3122和H2228进行检测,还检测了具有BCR-ABL融合基因的慢性白血病细胞K562,在其外泌体中也可以成功检测到BCR-ABL融合基因。通过对以上三种细胞的研究,证实融合基因的mRNA通过外泌体由胞内分泌到胞外是一种普遍现象,这一点为使用外泌体作为靶标进行临床检测奠定了基础。通过RT-PCR、IHC和Western Blot对89例NSCLC患者的肿瘤组织的EML4-ALK融合基因进行了检测,共筛选出15例具有融合基因的患者,其中V1融合形式5例,V2融合形式1例,V3融合形式8例和V5融合形式1例。收集这15例组织学检测为阳性患者的血清,分离提取血清中的外泌体,利用RT-PCR方法,对这15例患者的血清外泌体进行了EML4-ALK融合基因的检测。其中14例患者的血清外泌体中检测到EML4-ALK融合基因的存在,并且通过分析,发现血清外泌体中检测到的融合基因的融合形式与相应肿瘤组织中检测到的融合形式是一致的。而在组织学检测为阴性的74名患者中,同样以外泌体为靶标,进行融合基因的检测,检测到4例阳性患者。对这4例患者进一步扩大组织学检查范围,IHC结果显示为ALK阳性。该试验说明利用血清外泌体进行的检测不仅是无创的,而且还能够克服肿瘤异质性问题,提高了对EML4-ALK融合基因检测的准确度。由于这89名患者均属于癌症晚期病人,对这些血清标本进行早期诊断的研究,没有实际意义。因此,拟通过建立肺癌细胞裸鼠荷瘤模型,研究以外泌体为靶标进行肿瘤早期诊断的的可能性。将状态良好的H3122细胞、H2228细胞和A549细胞分别接种于4-6周龄的雌性裸鼠背部皮下,接种的细胞量为每只裸鼠接种1×106个细胞。接种后,定期测量裸鼠背部的肿瘤体积。H3122、H2228实验组每次每组取4只小鼠,A549对照组每组取1只进行眼球取血,分离血清外泌体,进行EML-ALK融合基因检测。实验结果显示,接种H3122细胞的裸鼠,在接种后的第4天才能够测量肿瘤体积的大小,但是在接种后的第2天,就可以在其血清外泌体中检测到EML4-ALK融合基因的存在。接种H2228细胞的裸鼠,在肿瘤形成的早期也可以在血清外泌体中检测到EML4-ALK融合基因的存在。裸鼠荷瘤的试验为以血清外泌体为靶标,对EML4-ALK融合基因早期检测的相关研究提供了一定的实验基础。综上所述,本课题首次证实外泌体中存在来源于肿瘤细胞的融合基因mRNA;肿瘤患者的血清外泌体可以检测到EML4-ALK融合基因,并且这种检测不仅可以做到对融合基因的确认,还可以确定融合类型。本课题为利用血清外泌体检测融合基因这一新的检测方法的建立以及靶向药的使用提供了一定的实验室参考依据。
[Abstract]:In the study of anaplastic large cell lymphoma, researchers first discovered the anaplastic lymphoma kinase (Anaplastic lymphoma, kinase, ALK). There is a tyrosine kinase receptor (Receptor tyrosine, kinase, RTKs). The chromosome inversion occurs, ALK gene may be related to echinoderm microtubule protein 4 (animal Echinoderm microtubule-associated protein-like 4, EML4) gene fusion, the formation of the EML4-ALK fusion gene.ALK gene with EML4 promoter, ALK kinase activation, lead to cancer, promote the occurrence and development of cancer. The study found that lung cancer may include a variety of solid tumors detected EML4-ALK fusion gene, among them, in non-small cell lung cancer (non-small cell lung cancer, NSCLC) in the highest detection rate, and the patients with distinctive clinical features, suggesting that the target is The molecular markers of lung adenocarcinoma with high specificity. At present, there have been at least 14 variants of EML4-ALK types, all of these expression products of fusion gene as a chimeric tyrosine kinase, has kinase activity, catalytic region can activate the ALK protein in the cytoplasm, activation of related signal transduction, promote cell proliferation and survival. The migration, so as to achieve the effect of cancer cells. Therefore, rapid, accurate and early detection of early detection of lung cancer EML4-ALK fusion gene, molecular typing and targeted therapy is very important. At present, all of the EML4-ALK fusion gene detection methods are based on the material after surgery or biopsy of the tumor tissue, unable to achieve early detection and the real-time monitoring, and because of tumor heterogeneity, makes the patient's test results may appear false negative. In recent years, liquid biopsy provides a new way for tumor detection, can effectively To avoid the limitations of biopsy. Exosome is one of the main material liquid biopsy detection. It formed: the first is the formation of early endosomes by cells through endocytosis, and early endosomal invagination after development of multivesicular body, when in the vicinity of the multivesicular body transferred to the cell membrane. With the cell membrane fusion, which originally exist in the multivesicular endosomes vesicles released into extracellular extracellular vesicles called exosomes by this way. Due to the formation process of exosome special, which can reflect the physiological state of cells containing secreted protein, and DNA microRNA and other signal molecules may contain information related to lesion. Cells that exosomes can provide potential biomarkers become rich target molecular source. Different diseases, exosomes lesion cells excreted in the water Some specific components. This makes different from body fluids (including blood, urine, pleural effusion and ascites) in the separation and purification of exosomes, then the molecular composition and the signal analysis possible, is expected to be used in disease diagnosis and monitoring, especially in the detection of tumor markers. Therefore, exosomes are likely to replace the organization biopsy under certain conditions, become a new non-invasive detection of disease targets, and can obtain information related to the early development of the disease. At present, a signal molecule in exosomes derived from tumor cells are reported: protein, double stranded DNA, microRNA and so on. But whether exosomes containing the fusion gene mRNA has not yet been reported. At present, with the fusion gene cell line three, respectively H3122, H2228 and DFCI032 cells. H3122 cells were H2228 cells, and does not have the EML4-ALK fusion gene in A549 cells as a negative control. For the test, to verify the feasibility of using exosomes detection of mRNA fusion gene. Firstly find the gene sequence of EML4 and ALK in NCBI, and according to the two different forms of fusion gene primers were designed. The extraction of H3122, A549 cells RNA by reverse transcriptase PCR, sequencing results confirmed that H3122 cell fusion site in EML4 exon thirteenth and exon twentieth of ALK fused with V1 fusion form. At the same time, the extraction of this two kinds of cell proteins were Western Blot experiments showed that in order to detect the expression of ALK protein and.Western Blot results, H3122 cells have a specific band at 120kD. Based on the analysis of EML4-ALK fusion protein and its size, found in the form of V1 fusion protein with the size of H3122. A549 cells, starved 24-48 hours later, supernatants were collected from cell culture supernatant Exosomes. Through the observation and analysis of morphology and exosome specific surface membrane protein, confirmed that exosomes successfully extracted. Extraction of exosomes in RNA, RT-PCR, the culture supernatant of exosomes can be successfully amplified to EML4-ALK fusion gene in H3122 cells, the sequencing results confirmed the fusion form is V1 that is consistent with the H3122 cell fusion form. Detection of EML4-ALK fusion gene and its fusion form using the same experimental method of H2228 cells. The sequencing results confirmed that the fusion form of V3a (fusion loci of EML4 gene exon sixth in twentieth and ALK gene exon V3b (fusion) and fusion site EML4 gene exon sixth 33bp intron and ALK gene with twentieth exons), fusion supernatants of exosomes can also be detected EML4-ALK fusion gene in H2228 cells, the cell fusion and form The form is the same. In addition to the detection of H3122 and H2228 lung cancer cells was also detected with chronic leukemia cells K562 BCR-ABL fusion gene, the outer urinary body can be successfully detected BCR-ABL fusion gene. Through the study of more than three kinds of cells, the fusion gene of mRNA confirmed by exosomes from cell to endocrine extracellular is a common phenomenon, which is the use of exosomes as targets for the clinical detection of the foundation. Through the RT-PCR, IHC and Western Blot in tumor tissues of 89 cases of patients with NSCLC EML4-ALK fusion gene were detected, 15 cases were screened with fusion gene with the V1 fusion form 5 cases of V2 fusion in 1 cases, V3 8 cases and V5 fusion fusion in 1 cases. 15 cases were collected for histological examination of the serum positive patients, extraction and separation of exosomes in serum, using RT-PCR method, 15 patients in this The serum exosomes were detected. The EML4-ALK fusion gene in serum of exosomes in 14 patients were detected EML4-ALK fusion gene, and through analysis, found that the fusion of form and corresponding tumor tissue fusion serum exosomes were detected in the gene was detected in the form of fusion is consistent. In histological examination of 74 patients were negative in the same body outside the urinary target detection of fusion gene, detected 4 positive cases. The scope of examination of the 4 cases of patients with further expansion of organization, IHC showed positive for ALK. The experiment shows that detection using serum exosomes were not only it is noninvasive, but also can overcome the problem of tumor heterogeneity, improves the efficiency of EML4-ALK fusion gene detection accuracy. Because of the 89 patients belong to patients with advanced cancer, research on early diagnosis of these serum samples, There is no practical significance. Therefore, we plan to establish tumor bearing nude mouse model of lung cancer cells, study of urinary body target for early diagnosis of cancer. The possibility of the good condition of H3122 cells, H2228 cells and A549 cells were cultured in a 4-6 week old female nude mice subcutaneously inoculated cells per mice inoculated with 1 * 106 cells. After inoculation, the tumor volume.H3122 regular measurement of the back of nude mice, H2228 experimental group, each group 4 mice in each group, A549 control group only 1 eye blood, serum separation of exosomes, EML-ALK fusion gene detection. Experiment results showed that the inoculation of H3122 cells into nude mice to measure tumor the size of the fourth days after inoculation, but on the second day after inoculation, it is possible to detect the EML4-ALK fusion gene in the serum of exosomes. H2228 cells were inoculated in nude mice, tumor formation in the early Can also detect the EML4-ALK fusion gene in serum of exosomes in tumor bearing nude mice. Tests for serum exosomes as a target, and provides an experimental basis for early detection of EML4-ALK fusion gene. To sum up, this paper first confirmed that the fusion gene mRNA in exosomes derived from tumor cell; serum exosomes in tumor patients can be detected and the detection of EML4-ALK fusion gene, not only can be done to confirm the fusion gene, can also determine the fusion type. The subject of this new fusion gene detection method for the detection of exosomes by serum as well as the establishment of targeted drug use will provide laboratory reference.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.43

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