本文選題：胃癌和癌前病變 + 胃多階段癌變模型��； 參考：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文

【摘要】：該博士論文由相對獨立的兩部分工作組成,其中第一部分分為二章。第一部分胃癌變相關(guān)分子事件研究第一章胃癌前病變及胃癌全基因組表達譜研究背景:胃癌變是一個多階段、多因素、多基因參與的復(fù)雜過程。胃多階段癌變模型認為正常胃粘膜進展為胃癌需要以下幾個連續(xù)階段:正常胃粘膜→非萎縮性胃炎→多灶萎縮性胃炎→腸化生→低級別上皮內(nèi)瘤變→高級別上皮內(nèi)瘤變→癌。利用表達譜芯片技術(shù)全面系統(tǒng)的對胃癌前病變及胃癌序列進行分子生物學(xué)研究,具有重要的臨床和科研價值。目的:本課題旨在研究胃癌變各階段組織在分子表達譜水平上的相似性和差異性;篩選胃癌變過程中可能起關(guān)鍵作用的分子,挖掘胃癌早期診斷標(biāo)志物。材料和方法:本部分研究收集了 66對胃不同病變階段新鮮組織,包括20例低級別上皮內(nèi)瘤變、23例高級別上皮內(nèi)瘤變、19例早期胃癌、4例進展期胃癌和配對的鄰近染色正常組織,用于全基因組表達譜芯片的檢測;收集了 50例胃癌患者的福爾馬林浸泡石蠟包埋組織樣本,一張切片上同時包括正常組織、癌前病變組織和胃癌組織,利用免疫組織化學(xué)染色(immunochemistry,IHC)對表達譜芯片數(shù)據(jù)篩選出的分子進行驗證。結(jié)果:胃癌前病變及胃癌樣本表達譜數(shù)據(jù)分析結(jié)果表明,在分子水平上胃癌前病變與胃癌具有相似性:很多腫瘤細胞的特征,例如DNA復(fù)制、細胞分裂、細胞周期等相關(guān)基因的上調(diào)以及生長負調(diào)控、支鏈氨基酸代謝等相關(guān)基因的下調(diào),在癌前病變階段已經(jīng)顯現(xiàn);癌前病變組織和胃癌組織相對于鄰近組織有著相似的差異表達模式,在胃癌組織中差異表達的基因絕大多在癌前病變階段已經(jīng)有了一致的差異表達。此外,胃癌前病變和胃癌在分子表達譜上存在差異性,主要涉及免疫反應(yīng)和細胞粘附。一方面,我們發(fā)現(xiàn)562個基因可能參與了胃癌變過程,其中在蛋白互作網(wǎng)絡(luò)中最為核心且緊密聯(lián)系的8-基因模塊為CD40LG、CTLA4、GZMB、ICAM1、ITGAX、SELL、TGFB1、TNF,這些分子參與了淋巴細胞和單核細胞的遷移、免疫負調(diào)控、細胞外基質(zhì)組織等過程,與胃癌患者的總生存相關(guān)。另一方面,為了尋找胃癌早期診斷標(biāo)志物,我們挑選了 4個在胃癌變過程中表達水平持續(xù)升高的基因進行后續(xù)免疫組化驗證,結(jié)果顯示,在胃癌變過程中,CCL3和CD300A的蛋白表達量和陽性率持續(xù)升高;與鄰近組織相比,OSM和CHI3L1在癌前病變組織和胃癌組織中的表達量和陽性率顯著升高,然而OSM和CHI3L1在癌前病變組織和胃癌組織中的表達量和陽性率沒有顯著差異。受試者工作特征曲線分析結(jié)果表明這四個分子對胃癌早期診斷可能具有一定的臨床價值。結(jié)論:在分子水平上胃癌前病變與胃癌具有相似性,胃癌前病變可能是胃癌發(fā)生的基礎(chǔ)。胃癌前病變和胃癌在分子表達譜上存在差異性,主要涉及免疫反應(yīng)和細胞粘附,這些基因的變化可能與癌前病變向胃癌惡性轉(zhuǎn)化密切相關(guān)。8-基因模塊可能在胃癌變中發(fā)揮關(guān)鍵作用。CCL3,CD300A,CHI3L1和OSM是潛在的胃癌早期診斷標(biāo)志物。以胃多階段癌變模型作為理論基礎(chǔ),利用胃不同病變階段樣本對胃癌變過程進行研究,有助于闡明胃癌發(fā)生發(fā)展機制、尋找癌變關(guān)鍵分子和潛在的胃癌早期診斷標(biāo)志物。第二章胃癌前病變及胃癌TCR免疫組庫研究背景:基于腫瘤浸潤淋巴細胞的免疫治療,主要依賴于抗原和T細胞受體(T-cell receptor,TCR)的相互作用,已經(jīng)在實體瘤的治療中顯現(xiàn)出潛在的臨床應(yīng)用價值。然而,TCR免疫組庫的很多基本特征仍然不清楚;癌變過程中TCR免疫組庫的變化,尤其是癌前病變階段,目前還沒有任何相關(guān)研究。目的:本課題旨在探究胃癌變過程中組織浸潤TCR免疫組庫的特征以及病變組織分子表型與病變微環(huán)境TCR免疫組庫的關(guān)系。材料和方法:研究納入了 41例不同病變階段的胃組織樣本,包括低級別上皮內(nèi)瘤變、高級別上皮內(nèi)瘤變、早期胃癌和配對的鄰近組織。我們通過多重PCR捕獲建庫和IluminaHiseq2000測序平臺對TCRβ互補決定區(qū)3進行了深度測序。此外,為了研究病變組織分子表型與病變微環(huán)境TCR免疫組庫的關(guān)系,我們對相應(yīng)的胃癌前病變樣本和早期胃癌樣本進行了全基因組表達譜芯片檢測。結(jié)果:分析結(jié)果顯示,T細胞克隆多樣性和TCR免疫組庫的頻率分布在胃癌變過程中沒有顯著變化。然而,在胃癌變過程中,病變組織和配對鄰近組織TCR免疫組庫的相似性逐漸降低。病變組織和配對鄰近組織之間的TCR免疫組庫的重疊逐漸降低,為抗原驅(qū)動克隆擴增提供了直接的證據(jù);越來越多的病變抗原相關(guān)T細胞克隆變成了高頻克隆,而且它們占有的頻率在胃癌變過程中逐漸增加。此外,病變組織和配對鄰近組織之間的共有高頻T細胞克隆的頻率相似性在胃癌變不同階段迅速降低。最后,利用基于網(wǎng)絡(luò)的派系過濾方法對表達譜芯片數(shù)據(jù)和TCR免疫組庫變異指數(shù)進行整合分析,我們找到了一個包含11個基因的模塊,這11個基因的表達量與胃癌病人的總生存顯著相關(guān)。結(jié)論:我們的研究闡明了在胃癌變過程中組織浸潤TCR免疫組庫的多階段異質(zhì)性。突變細胞和免疫系統(tǒng)之間的相互作用可能在細胞惡性轉(zhuǎn)化過程中發(fā)揮重要作用。本研究加深了我們對胃癌變過程中免疫反應(yīng)的理解。第二部分SMC4在肺腺癌中的功能及相關(guān)分子機制研究背景:越來越多的證據(jù)顯示,胚胎發(fā)育和腫瘤形成在組織形態(tài)、細胞生物學(xué)學(xué)行為以及分子表達模式等方面具有極大的相似性。利用胚胎發(fā)育與腫瘤發(fā)生的相似性,重新審視正常發(fā)育的獨特過程,深入挖掘胚胎發(fā)育過程中分子譜的變化規(guī)律,可以在最大程度上規(guī)避腫瘤異質(zhì)性給研究帶來的種種困難。染色體結(jié)構(gòu)維持蛋白4(Structural maintenance of chromosome 4,SMC4)是集縮素蛋白復(fù)合體(condensin)的核心蛋白,主要參與染色體的凝集和分離。實驗室前期發(fā)表的人胚胎肺發(fā)育樣本和肺腺癌樣本mRNA表達譜數(shù)據(jù)分析結(jié)果顯示,SMC4在胚胎肺發(fā)育過程中持續(xù)下調(diào),在肺腺癌中卻呈現(xiàn)高表達。目的:本課題旨在探索從肺發(fā)育數(shù)據(jù)中挖掘的蛋白SMC4在肺腺癌發(fā)生發(fā)展中所發(fā)揮的功能及相關(guān)分子機制。材料和方法:首先,利用實時定量PCR(Real-timePCR)對43對手術(shù)切除的肺腺癌組織及其癌旁組織的SMC4 mRNA表達水平進行驗證。接著,利用IHC對75例FFPE肺腺癌組織及其癌旁組織的SMC4蛋白表達水平進行了驗證,并利用log-rank檢驗分析了 SMC4表達量與肺腺癌病人生存的關(guān)系。利用CCK-8實驗和transwell實驗檢測SMC4對肺腺癌細胞增殖和侵襲的影響。為了進一步探索SMC4在肺腺癌發(fā)生發(fā)展中的機制,我們通過免疫共沉淀聯(lián)合質(zhì)譜分析找到了 SMC4潛在的相互作用蛋白DDX46(DEAD-boxhelicase46)。最后,利用免疫共沉淀和免疫熒光共定位實驗驗證SMC4和DDX46的相互作用。結(jié)果:Real-time PCR和IHC分別從RNA和蛋白水平上證實了SMC4在肺腺癌組織中的表達量顯著高于癌旁組織,而且SMC4蛋白高表達是肺腺癌獨立的預(yù)后不良因素(log-rank檢驗,p= 0.036)。細胞實驗表明,SMC4敲降顯著抑制A549細胞的增殖和侵襲。此外,SMC4敲降后,與增殖相關(guān)的蛋白PLK1,Cyclin B1,CDK1表達量顯著下調(diào)。對人胚胎肺發(fā)育樣本和肺腺癌樣本mRNA表達譜數(shù)據(jù)進行分析發(fā)現(xiàn),SMC4和DDX46表達模式相似;Speraman相關(guān)性分析結(jié)果表明,SMC4和DDX46的mRNA表達水平顯著正相關(guān)(r=0.629,p0.0001)。正反向Co-IP證實SMC4和DDX46存在相互作用。免疫熒光共定位實驗發(fā)現(xiàn),SMC4和DDX46在細胞周期的各個時相均存在共定位現(xiàn)象。結(jié)論:SMC4在肺腺癌組織中的表達量顯著高于癌旁肺組織,而且SMC4蛋白高表達是肺腺癌獨立的預(yù)后不良因素。SMC4促進肺腺癌細胞的增殖和侵襲。SMC4和DDX46存在相互作用,可能通過影響pre-mRNA剪切參與肺腺癌的發(fā)生。在發(fā)育過程中與SMC4有相似表達模式的基因,可能同時參與腫瘤的形成。通過胚胎發(fā)育相關(guān)分子事件對腫瘤發(fā)生進行比較學(xué)研究,是一種挖掘潛在的腫瘤標(biāo)志物和治療靶點的有效方法,也有助于闡明腫瘤的發(fā)生發(fā)展機制。
[Abstract]:The doctoral thesis is composed of two parts, which are divided into two chapters. The first part is divided into two chapters. Part 1: the first part of the study of gastric cancer related molecular events: Chapter 1 research background of the whole genome expression profiles of gastric precancerous lesions and gastric cancer: gastric cancer is a multi stage, multi factor, complex process of multi gene participation. The normal gastric mucosa progresses to the following stages: normal gastric mucosa, non atrophic gastritis, multifocal atrophic gastritis, intestinal metaplasia, low-grade intraepithelial neoplasia, high grade intraepithelial neoplasia and cancer. It has important clinical and scientific value. Objective: the purpose of this study is to study the similarity and difference in the level of molecular expression profiles at various stages of gastric cancer, to screen the molecules that may play a key role in the process of gastric cancer change, and to excavate early diagnosis markers for gastric cancer. Materials and methods: 66 pairs of different stages of gastric lesions were collected in this study. Fresh tissue, including 20 cases of low-grade intraepithelial neoplasia, 23 cases of high grade intraepithelial neoplasia, 19 cases of early gastric cancer, 4 cases of advanced gastric cancer and paired adjacent normal tissues, were used for the detection of whole genome expression chips; 50 cases of formalin were collected and paraffin embedded tissue samples were collected in 50 cases of gastric cancer. Often tissue, precancerous tissue and gastric cancer tissue, immunochemistry (IHC) was used to verify the molecules screened by the expression spectrum chip data. Results: the analysis of the expression profiles of precancerous lesions and gastric cancer samples showed that the precancerous lesions were similar to gastric cancer at the molecular level: many tumor cells. Characteristics, such as DNA replication, cell division, cell cycle related genes up-regulated and negative regulation of growth, and downregulation of related genes, such as branched chain amino acid metabolism, have appeared in the precancerous lesion stage; precancerous tissue and gastric cancer tissues have similar differential expression patterns relative to adjacent tissues, and are differentially expressed in gastric cancer tissues. In addition, precancerous lesions and gastric cancer are different in molecular expression profiles, mainly involving immune response and cell adhesion. On the one hand, we find that 562 genes may be involved in the process of gastric cancer, the most important and closely linked in the protein interaction network. The 8- gene module of the system is CD40LG, CTLA4, GZMB, ICAM1, ITGAX, SELL, TGFB1, TNF. These molecules participate in the migration of lymphocytes and mononuclear cells, the negative regulation of immunization, the extracellular matrix, and the total survival of the gastric cancer patients. On the other hand, we choose 4 to change the process of gastric cancer in order to find the early diagnosis marker of gastric cancer. The results showed that the protein expression and positive rate of CCL3 and CD300A increased continuously during the process of gastric cancer, and the expression and positive rate of OSM and CHI3L1 in precancerous and gastric cancer tissues were significantly higher than those of adjacent tissues, while OSM and CHI3L1 were in precancerous disease. There is no significant difference in the expression and positive rate of the tissue and gastric carcinoma. The results of the work characteristic curve of the subjects show that these four molecules may have certain clinical value for the early diagnosis of gastric cancer. Conclusion: the precancerous lesions are similar to gastric cancer at the molecular level. Precancerous lesions may be the basis of gastric cancer. Precancerous lesions and gastric cancer are different in molecular expression profiles, mainly involving immune response and cell adhesion. The changes of these genes may be closely related to the malignant transformation of precancerous lesions to gastric cancer. The.8- gene module may play a key role in gastric cancer..CCL3, CD300A, CHI3L1 and OSM are potential markers for the early diagnosis of gastric cancer. The multi-stage carcinogenesis model is a theoretical basis, using samples of different stages of gastric lesions to study the process of gastric cancer. It is helpful to elucidate the mechanism of gastric cancer and development, to find the key molecules of cancer and the potential diagnostic markers for the early gastric cancer. Second the background of the study on the precancerous lesions of gastric cancer and the TCR immunization group of gastric cancer: Based on tumor infiltrating lymphocytes Immunotherapy, which is mainly dependent on the interaction of antigen and T cell receptor (T-cell receptor, TCR), has shown potential clinical value in the treatment of solid tumors. However, many basic features of the TCR immune group are still unclear, and the changes in the TCR immune group, especially the precancerous phase of the cancerous process, are not yet available. Objective: To explore the characteristics of tissue infiltrating TCR immune group during gastric cancer and the relationship between the molecular phenotype of the pathological tissue and the TCR immune group Library in the pathological microenvironment. Materials and methods: the study included 41 samples of gastric tissue in different pathological stages, including low-grade intraepithelial neoplasia and high grade intraepithelial neoplasia. Tumorigenicity, early gastric cancer and paired adjacent tissues. We sequenced the TCR beta complementation area 3 by multiple PCR capture building and IluminaHiseq2000 sequencing. In addition, in order to study the relationship between the molecular phenotype of the lesion and the TCR immune group of the pathological microenvironment, we have the corresponding precancerous lesion samples and early gastric cancer samples. The results showed that the diversity of T cell clones and the frequency distribution of the TCR immune group did not change significantly in the process of gastric cancer. However, in the process of gastric cancer, the similarity between the pathological and the paired adjacent tissues of the TCR immune group gradually decreased. The pathological tissue and the paired adjacent groups were gradually reduced. The overlap between the TCR immune group libraries of the fabric decreased gradually and provided direct evidence for the antigen driven cloning and amplification, and more and more clones of the T cells were transformed into high frequency clones, and the frequency of their occupancy increased gradually in the process of gastric cancer. In addition, the common high frequency T cells between the lesion group and the adjacent adjacent tissues were found. The frequency similarity of the clones decreased rapidly at different stages of gastric cancer. Finally, a network based factional filtering method was used to integrate the expression spectrum chip data and the TCR immune group variation index. We found a module containing 11 genes. The amount of the 11 genes was significantly related to the total survival of the gastric cancer patients. Our study elucidated the multistage heterogeneity of the tissue infiltrating TCR immune group during the process of gastric cancer. The interaction between the mutant cells and the immune system may play an important role in the process of cell malignant transformation. This study deepens our understanding of the immune response in the process of gastric cancer change. The second part of the SMC4 is in the lung adenocarcinoma. Research background: more and more evidence shows that embryonic development and tumor formation have great similarity in tissue morphology, cell biological behavior and molecular expression patterns. Using the similarity of embryo development and tumor occurrence, it reexamines the unique process of normal development and deeply excavate the embryo. The variation of molecular spectrum during fetal development can avoid the difficulties caused by tumor heterogeneity to the maximum extent. Chromosome structure maintenance protein 4 (Structural maintenance of chromosome 4, SMC4) is the core egg white of the aggregation protein complex (condensin), which is mainly involved in the agglutination and separation of chromosomes. The results of human embryonic lung development samples and mRNA expression profiles of lung adenocarcinoma samples showed that SMC4 was down regulated during embryonic lung development and showed high expression in lung adenocarcinoma. Objective: the purpose of this study is to explore the function and related molecules of protein SMC4 in the development and development of lung adenocarcinoma from lung development data. Mechanism, materials and methods: first, using real-time quantitative PCR (Real-timePCR) to verify the expression level of SMC4 mRNA in 43 pairs of surgically resected lung adenocarcinoma tissues and adjacent tissues. Then, the expression of SMC4 protein in 75 cases of FFPE lung adenocarcinoma tissue and its para cancerous tissues was verified by IHC, and SMC4 was analyzed by log-rank test. The relationship between expression and survival of lung adenocarcinoma patients. The effects of SMC4 on proliferation and invasion of lung adenocarcinoma cells were detected by CCK-8 and Transwell experiments. In order to further explore the mechanism of SMC4 in the development of lung adenocarcinoma, we found the potential interaction protein DDX46 (DEAD-boxhelica) by immunoprecipitation combined with mass spectrometry analysis. Se46). Finally, the interaction between immunofluorescence and immunofluorescence was used to verify the interaction between SMC4 and DDX46. Results: Real-time PCR and IHC showed that the expression of SMC4 in lung adenocarcinoma tissues was significantly higher than that in the para cancerous tissues from RNA and protein levels, and the high expression of SMC4 protein was an independent prognostic factor for lung adenocarcinoma (log-rank). Test, p= 0.036). Cell experiments showed that SMC4 knockdown significantly inhibited the proliferation and invasion of A549 cells. In addition, after SMC4 knockdown, the expression of PLK1, Cyclin B1, CDK1 was significantly down after the SMC4 knockdown. The SMC4 and DDX46 expression patterns were found to be similar to the SMC4 and DDX46 expression patterns for the human fetal lung development samples and lung adenocarcinoma samples; The results of the correlation analysis showed that the mRNA expression level of SMC4 and DDX46 was positively correlated (r=0.629, P0.0001). The positive and reverse Co-IP confirmed the interaction between SMC4 and DDX46. The co localization of SMC4 and DDX46 in each phase of the cell cycle was found in the immunofluorescence co localization experiment. Conclusion: SMC4 in the lung adenocarcinoma tissues was significantly higher than that of SMC4. Para cancerous lung tissue, and high expression of SMC4 protein is an independent prognostic factor for lung adenocarcinoma,.SMC4 promotes the proliferation of lung adenocarcinoma cells and the interaction of.SMC4 and DDX46, and may be involved in the occurrence of lung adenocarcinoma by affecting pre-mRNA shear. In the process of development, the gene similar to SMC4 may be involved in the shape of the tumor at the same time. The comparative study of tumorigenesis through embryonic development related molecular events is an effective method for mining potential tumor markers and targets, and also helps to elucidate the mechanism of tumor development and development.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.2

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