本文選題：融合基因　切入點：EML4-ALK　出處：《中國人民解放軍軍事醫(yī)學科學院》2017年碩士論文

【摘要】：在對間變性大細胞淋巴瘤的研究中,研究者首次發(fā)現(xiàn)了間變性淋巴瘤激酶(Anaplastic lymphoma kinase,ALK)的存在。它是一種酪氨酸激酶受體(Receptor tyrosine kinase,RTKs)。由于染色體倒位的發(fā)生,ALK基因能夠與棘皮動物微管相關(guān)類蛋白4(Echinoderm microtubule-associated protein-like 4,EML4)基因發(fā)生融合,形成EML4-ALK融合基因。ALK基因通過借助EML4的啟動子,使ALK激酶活化,導(dǎo)致細胞發(fā)生癌變,促進癌癥的發(fā)生和發(fā)展。目前研究發(fā)現(xiàn),包括肺癌在內(nèi)的多種實體瘤中均可檢測到EML4-ALK融合基因的存在,其中,在非小細胞肺癌(non-small cell lung cancer,NSCLC)中的檢出率最高,并且該類患者具有鮮明的臨床特征,提示該靶點是肺腺癌特異性較高的分子標記物。目前,至少發(fā)現(xiàn)了14種EML4-ALK的變異體類型,所有這些融合基因的表達產(chǎn)物為一種嵌合型酪氨酸激酶,具有激酶活性,可激活A(yù)LK蛋白的胞內(nèi)催化區(qū)域,激活相關(guān)信號通路,促進細胞增殖、存活、遷移,從而達到細胞癌變的效果。因此,快速、準確和及早地檢測EML4-ALK融合基因?qū)Ψ伟┑脑缙跈z測、分子分型和靶向治療至關(guān)重要。目前,所有EML4-ALK融合基因的檢測方法都是針對手術(shù)或者活檢取材后的腫瘤組織進行的,無法實現(xiàn)早期檢測和實時監(jiān)測,且由于腫瘤異質(zhì)性問題,使得患者的檢測結(jié)果可能出現(xiàn)假陰性。近年來,液態(tài)活檢為腫瘤的檢測提供新的思路,能夠有效的避免組織活檢的局限性。外泌體是液態(tài)活檢主要檢測的物質(zhì)之一。它的形成過程:首先是由細胞通過內(nèi)吞作用形成早期內(nèi)體,而后早期內(nèi)體經(jīng)過內(nèi)陷發(fā)展為多囊泡內(nèi)體,當多囊泡內(nèi)體轉(zhuǎn)移至細胞膜附近時,其與細胞膜發(fā)生融合,從而導(dǎo)致原本存在于多囊泡內(nèi)體中的囊泡釋放到細胞外,通過這種方式分泌到胞外的囊泡稱為外泌體。由于外泌體特殊的形成過程,使得其中含有能夠反映分泌細胞生理狀態(tài)的蛋白質(zhì)、DNA和microRNA等信號分子,可能包含了細胞病變相關(guān)的信息。這一點使外泌體成為能夠提供豐富的潛在生物標志物分子源的靶標。疾病的不同,病變細胞所分泌到胞外的外泌體中含有的特定成分不同。這使得從體液(包括血液、尿液、胸腹水等)中分離純化外泌體,再對其組成及信號分子進行分析成為可能,有望應(yīng)用于疾病診斷及監(jiān)測,尤其在腫瘤標志物檢測方面。因此,外泌體極有可能在一定條件下代替組織活檢,成為一種新型無創(chuàng)的疾病檢測靶標,并且可以獲得疾病早期發(fā)展的相關(guān)信息。目前,關(guān)于外泌體中信號分子的報道有:來源于腫瘤細胞的蛋白質(zhì)、雙鏈DNA、microRNA等。但外泌體中是否含有融合基因的mRNA還尚未報道。目前具有該融合基因的細胞系有三種,分別為H3122、H2228以及DFCI032細胞。選取H3122細胞、H2228細胞以及不具有EML4-ALK融合基因的A549細胞作為陰性對照進行試驗,對使用外泌體檢測融合基因mRNA的可行性進行初步驗證。首先在NCBI上查找EML4和ALK的基因序列,并針對這兩種基因的幾種不同融合形式分別設(shè)計引物。提取H3122、A549細胞的RNA進行逆轉(zhuǎn)錄PCR,測序結(jié)果證實H3122細胞發(fā)生融合的位點在EML4的第13外顯子與ALK的第20外顯子發(fā)生融合,即為V1融合形式。同時,提取這兩種細胞的蛋白質(zhì)進行Western Blot實驗以檢測ALK蛋白的表達情況。Western Blot實驗結(jié)果顯示,H3122細胞在120kD處有一條特異性條帶,通過對EML4-ALK融合蛋白大小進行分析,發(fā)現(xiàn)其與V1形式的融合蛋白大小相符。將H3122、A549細胞饑餓處理24-48小時后,收集細胞培養(yǎng)上清,提取細胞培養(yǎng)上清中的外泌體。通過電鏡形態(tài)學觀察以及外泌體特異性表面膜蛋白的分析,證實外泌體提取成功。提取外泌體中的RNA,進行RT-PCR,在H3122細胞培養(yǎng)上清外泌體可以成功擴增到EML4-ALK融合基因,測序結(jié)果證實其融合形式也是V1,與H3122細胞中的融合形式是一致的。對H2228細胞使用相同的實驗方法檢測EML4-ALK融合基因及其融合形式,測序結(jié)果證實其融合形式為V3a(融合位點為EML4基因的第6外顯子與ALK基因的第20外顯子發(fā)生融合)和V3b(融合位點為EML4基因的第6外顯子加上33bp內(nèi)含子與ALK基因的第20外顯子發(fā)生融合),在H2228細胞培養(yǎng)上清外泌體中也可以檢測到EML4-ALK融合基因,其融合形式與細胞的融合形式是一致的。除了對肺癌細胞H3122和H2228進行檢測,還檢測了具有BCR-ABL融合基因的慢性白血病細胞K562,在其外泌體中也可以成功檢測到BCR-ABL融合基因。通過對以上三種細胞的研究,證實融合基因的mRNA通過外泌體由胞內(nèi)分泌到胞外是一種普遍現(xiàn)象,這一點為使用外泌體作為靶標進行臨床檢測奠定了基礎(chǔ)。通過RT-PCR、IHC和Western Blot對89例NSCLC患者的腫瘤組織的EML4-ALK融合基因進行了檢測,共篩選出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。收集這15例組織學檢測為陽性患者的血清,分離提取血清中的外泌體,利用RT-PCR方法,對這15例患者的血清外泌體進行了EML4-ALK融合基因的檢測。其中14例患者的血清外泌體中檢測到EML4-ALK融合基因的存在,并且通過分析,發(fā)現(xiàn)血清外泌體中檢測到的融合基因的融合形式與相應(yīng)腫瘤組織中檢測到的融合形式是一致的。而在組織學檢測為陰性的74名患者中,同樣以外泌體為靶標,進行融合基因的檢測,檢測到4例陽性患者。對這4例患者進一步擴大組織學檢查范圍,IHC結(jié)果顯示為ALK陽性。該試驗說明利用血清外泌體進行的檢測不僅是無創(chuàng)的,而且還能夠克服腫瘤異質(zhì)性問題,提高了對EML4-ALK融合基因檢測的準確度。由于這89名患者均屬于癌癥晚期病人,對這些血清標本進行早期診斷的研究,沒有實際意義。因此,擬通過建立肺癌細胞裸鼠荷瘤模型,研究以外泌體為靶標進行腫瘤早期診斷的的可能性。將狀態(tài)良好的H3122細胞、H2228細胞和A549細胞分別接種于4-6周齡的雌性裸鼠背部皮下,接種的細胞量為每只裸鼠接種1×106個細胞。接種后,定期測量裸鼠背部的腫瘤體積。H3122、H2228實驗組每次每組取4只小鼠,A549對照組每組取1只進行眼球取血,分離血清外泌體,進行EML-ALK融合基因檢測。實驗結(jié)果顯示,接種H3122細胞的裸鼠,在接種后的第4天才能夠測量腫瘤體積的大小,但是在接種后的第2天,就可以在其血清外泌體中檢測到EML4-ALK融合基因的存在。接種H2228細胞的裸鼠,在腫瘤形成的早期也可以在血清外泌體中檢測到EML4-ALK融合基因的存在。裸鼠荷瘤的試驗為以血清外泌體為靶標,對EML4-ALK融合基因早期檢測的相關(guān)研究提供了一定的實驗基礎(chǔ)。綜上所述,本課題首次證實外泌體中存在來源于腫瘤細胞的融合基因mRNA;腫瘤患者的血清外泌體可以檢測到EML4-ALK融合基因,并且這種檢測不僅可以做到對融合基因的確認,還可以確定融合類型。本課題為利用血清外泌體檢測融合基因這一新的檢測方法的建立以及靶向藥的使用提供了一定的實驗室參考依據(jù)。
[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.

【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R730.43

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