本文選題：基因芯片 + 差異表達基因　； 參考：《天津醫(yī)科大學》2017年碩士論文

【摘要】：背景:卵巢癌(Ovarian carcinoma,OC)屬女性三大惡性腫瘤,雖發(fā)病率小于宮頸癌和宮體癌,但卻是女性生殖系統(tǒng)惡性程度最高的癌癥。卵巢癌患病時早期癥狀不明顯,且缺乏可靠的早期診斷手段,約有70%的患者在確診時已進入到疾病進展期。卵巢癌的早期診斷,是關系到病人預后的最重要的因素�；蛐酒夹g已成為卵巢癌的早期診斷方法之一。外周血單個核細胞(peripheral blood mononuclear cells,PBMC)除淋巴細胞和單核細胞外,也包含一些循環(huán)腫瘤細胞(circulating tumor cells,CTCs)和腫瘤干細胞(tumor stem cells,TSCs),在腫瘤病患中的改變可以體現(xiàn)機體的微環(huán)境變化。本文利用基因芯片技術檢測卵巢癌患者和健康人對照的外周血中,PBMC基因表達譜的差異,不僅有利于卵巢癌的早期診斷,且還可能揭示卵巢癌發(fā)病、轉移中的機制。單鏈抗體(single-chain fragment variable,sc Fv)是由抗體的重鏈和輕鏈的可變區(qū)經(jīng)過一段15~20個氨基酸的片段連接而成,是有功能抗體的最小的形式。單鏈抗體的相對分子質(zhì)量小,組織穿透能力強,免疫原性低,在腫瘤的診斷和治療上有著廣泛的應用,如將單鏈抗體連接放射性標記物,應用于腫瘤組織的原位成像,或連接抗腫瘤的細胞毒素或化療藥物,進行腫瘤的治療等。本文利用酵母展示技術,成功構建了卵巢癌患者的單鏈抗體庫,并用卵巢癌A2780細胞株對抗體庫進行了初步篩選,為今后進一步獲取可以應用于臨床的單鏈抗體提供了基礎。目的:一、應用基因芯片技術檢測卵巢癌患者和健康人對照的外周血中,PBMC的基因表達譜差異,篩選卵巢癌患者差異表達基因(differentially expressed genes,DEG),對差異基因進行基因通路(pathway)分析、基因本體論(GO)分析和功能分析。二、構建卵巢癌患者的酵母展示單鏈抗體庫,對構建的單鏈抗體庫測定庫容進行初步評價,并用卵巢癌細胞株A2780對抗體庫進行初步篩選,為進一步獲取高親和力的卵巢癌單鏈抗體提供基礎。方法:一、提取臨床確診為卵巢癌的8例患者和4例健康女性的外周血中的PBMC樣本,提取PBMC中的總RNA,將卵巢癌患者的樣本兩兩混合成四份樣本,以減少樣本間差異。利用商業(yè)化基因芯片Affymetrix?Human Genome U219 Array Strip對兩組樣本進行轉錄本基因表達量的檢測,利用軟件和在線網(wǎng)站對結果進行分析。二、取13例卵巢癌患者外周血分離PBMC,TRIzol法提取PBMC總RNA,逆轉錄PCR合成出cDNA,利用設計的引物擴增出全套輕鏈可變區(qū)(VL)和重鏈可變區(qū)(VH)基因片段。使用重疊延伸PCR(SOE-PCR)將輕、重鏈基因片段連接。利用真核細胞體內(nèi)同源重組系統(tǒng)介導的間隙修復(gap-repair)機制,將連接好的單鏈抗體片段和線性化的質(zhì)粒p YD1同時轉入酵母菌中。使用不含色氨酸的篩選培養(yǎng)基進行轉化后的篩選。在半乳糖存在的誘導培養(yǎng)基中,酵母將單鏈抗體表達于酵母細胞表面,建立卵巢癌患者的單鏈抗體庫。而后使用卵巢癌細胞株A2780與表達單鏈抗體的酵母細胞進行結合,利用流式細胞術對可結合A2780細胞的酵母菌株進行初步篩選。結果:一、基因芯片檢測后分析發(fā)現(xiàn),卵巢癌患者PBMC相比健康人PBMC中差異表達基因共1158個(Fold Change絕對值2倍,p0.05),其中上調(diào)的差異表達基因有311個,下調(diào)的差異表達基因847個。上調(diào)差異最大的5個基因是THBS1、NR4A2、BTG1、ADM、MIR22,下調(diào)差異最大的5個基因是GIMAP8、CX3CR1、GIMAP4、ST8SIA4、MYOM2。對差異基因進行通路分析后發(fā)現(xiàn),涉及的通路主要分布在信號轉導系統(tǒng)和免疫系統(tǒng)。對差異基因進行GO功能富集分析后發(fā)現(xiàn),差異基因在細胞組成分析中,主要涉及細胞組分和細胞器,在分子功能分析中主要具有結合和催化活性,同時在生物學過程分析中,主要對細胞過程和代謝過程起作用。對差異基因進行功能分析后,在分類為凋亡的基因與GO分析為免疫系統(tǒng)過程的結果取交集,共篩選出有意義的差異基因6個。二、利用PCR方法成功擴增出部分VH、VL基因片段,VH、VL連接后和線性化的質(zhì)粒共同轉化進入酵母菌中,在篩選培養(yǎng)基中有菌落生長,表明轉化成功。對建立的單鏈抗體庫進行了庫容的檢測,測定庫容為:κ鏈抗體庫庫容為4×109、λ鏈抗體庫庫容為8×109。使用卵巢癌細胞系A2780對抗體庫進行了篩選,共得到展示VH-VLκ鏈陽性的酵母菌株49株,展示VH-VLλ鏈陽性酵母菌株90株。結論:一、卵巢癌患者和健康對照人群的PBMC中基因表達譜存在顯著差異,篩選的卵巢癌患者差異基因為進一步研究卵巢癌提供了基礎。二、利用酵母表面展示系統(tǒng)成功構建出卵巢癌的單鏈抗體庫,并在誘導下在酵母細胞表面表達抗卵巢癌細胞的單鏈抗體,利用卵巢癌腫瘤細胞系A2780對抗體庫進行了初步篩選,為以后進一步獲取高親和力的卵巢癌單鏈抗體提供了基礎。
[Abstract]:Background: Ovarian carcinoma (OC) is a female three major malignant tumor. Although the incidence is less than the cervical and uterine cancer, it is the most malignant cancer in the female reproductive system. The early symptoms of ovarian cancer are not obvious, and the early diagnosis method is lack, and about 70% of the patients have entered the stage of disease progression. The early diagnosis of nesting cancer is the most important factor in the prognosis of patients. Gene chip technology has become one of the early diagnostic methods for ovarian cancer. Peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMC) also contain a number of circulating tumor cells (circulating tumor cells, CTCs) except for lymphocytes and mononuclear cells. The change of tumor stem cells (TSCs) in the cancer patients can reflect the changes in the microenvironment of the body. In this paper, the differential expression of PBMC gene expression in the peripheral blood of the ovarian cancer patients and the healthy people is detected by gene chip technology, which is not only beneficial to the early diagnosis of ovarian cancer, but also may reveal the pathogenesis and metastasis of ovarian cancer. The mechanism. Single-chain fragment variable (SC Fv) is the smallest form of functional antibody with a fragment of the variable region of the antibody's heavy chain and light chain through a segment of 15~20 amino acids. The relative molecular weight of the single chain antibody is small, the tissue penetrates the energy, the immunogenicity is low, and the tumor is diagnosed and treated. It has a wide range of applications, such as the use of single chain antibody to connect radioactive markers, in situ imaging of tumor tissue, or to connect antitumor cytotoxin or chemotherapeutic drugs, and to treat cancer. In this paper, the single chain antibody library of ovarian cancer patients was successfully constructed by yeast display technology, and the antibody library was used for ovarian cancer A2780 cell line. Preliminary screening is carried out to provide a basis for further obtaining the single chain antibody that can be applied to the clinic in the future. Objective: to detect the difference in gene expression profiles of PBMC in peripheral blood of ovarian cancer patients and healthy people, and to screen the differential expression genes (differentially expressed genes, DEG) of ovarian cancer patients, and the difference between them. The gene pathway (pathway) analysis, Gene Ontology (GO) analysis and functional analysis. Two, the yeast display single chain antibody library for ovarian cancer patients was constructed to evaluate the capacity of the single chain antibody library, and the ovarian cancer cell line A2780 was used to screen the body library to further obtain the high affinity of the ovary. Methods: 1. Methods: first, PBMC samples from 8 patients with ovarian cancer and 4 healthy women were extracted from the peripheral blood of 4 healthy women, and the total RNA in PBMC was extracted, and the sample 22 of the ovarian cancer patients was mixed into four samples to reduce the difference between samples. Commercial gene chip Affymetrix? Human Genome U219 Array Stri was used. P was used to detect the expression of the transcriptional gene in the two groups, and the results were analyzed by software and online website. Two, the peripheral blood of 13 patients with ovarian cancer was isolated from the peripheral blood PBMC, the PBMC total RNA was extracted by TRIzol, and cDNA was synthesized by reverse transcriptase PCR. The whole set of light chain variable region (VL) and the heavy chain variable region (VH) gene fragment were amplified by the designed primers. Using overlapping extension PCR (SOE-PCR) to connect light and heavy chain gene fragments. Using the mechanism of gap repair (gap-repair) mediated by homologous recombination system in eukaryotic cells, the linked single chain antibody fragment and linearized plasmid P YD1 are transferred to yeast simultaneously. The screened medium without tryptophan is used for the transformation after screening. In the inducible medium of sugar, the yeast expressed the single chain antibody to the yeast cell surface and established the single chain antibody library of the ovarian cancer patients. Then, the yeast cells that expressed single chain antibody were used to combine the ovarian cancer cell line A2780, and the yeast strain which could be combined with A2780 cells was screened by flow cytometry. After the analysis of the microchip detection, there were 1158 differentially expressed genes in the ovarian cancer patients' PBMC compared with the healthy PBMC (Fold Change absolute value 2 times, P0.05), in which there were 311 differentially expressed genes up and 847 differentially expressed genes. The 5 most differentially regulated genes were THBS1, NR4A2, BTG1, ADM, MIR22, and the 5 most differentially differentially regulated genes. GIMAP8, CX3CR1, GIMAP4, ST8SIA4, MYOM2. showed that the pathway involved in the differential gene was mainly distributed in the signal transduction system and the immune system. After the GO enrichment analysis of the differential genes, the differential genes were found in the cell composition analysis, mainly involved in the cell components and organelles, and in the molecular functional analysis. We should have binding and catalytic activity, and play a role in cellular process and metabolic process in biological process analysis. After functional analysis of the differential genes, we have intersected the results of the classified as apoptotic genes and GO analysis for the immune system process. A total of 6 significant differential genes were screened. Two, a successful amplification by PCR method. Some VH, VL gene fragments, VH, VL were joined together with the linearized plasmid into the yeast, and the colony growth was found in the screening medium, which showed that the transformation was successful. The storage capacity of the single chain antibody library was detected, the capacity of the kappa chain antibody library was 4 * 109, and the lambda chain antibody library capacity was 8 * 109. using ovarian cancer cells. The antibody library was screened by A2780. A total of 49 strains of yeast strains showing VH-VL kappa chain positive were obtained and 90 strains of VH-VL lambda positive yeast strain were displayed. Conclusion: first, the gene expression profiles in the PBMC of the ovarian cancer patients and the healthy controls were significantly different, and the differential genes of the selected ovarian cancer patients provided the basis for further study of ovarian cancer. Two, the single chain antibody library of ovarian cancer was successfully constructed by the yeast surface display system, and the single chain antibody against ovarian cancer cells was expressed on the surface of the yeast cells. The antibody library was preliminarily screened by the ovarian cancer cell line A2780, which provided a basis for further obtaining the high affinity ovarian cancer single chain antibody.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.31

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本文編號：1850342