卵巢癌患者的PBMC差异表达基因筛选及卵巢癌单链抗体库的构建

发布时间：2018-05-06 02:19

本文选题：基因芯片 + 差异表达基因　；参考：《天津医科大学》2017年硕士论文

【摘要】：背景:卵巢癌(Ovarian carcinoma,OC)属女性三大恶性肿瘤,虽发病率小于宫颈癌和宫体癌,但却是女性生殖系统恶性程度最高的癌症。卵巢癌患病时早期症状不明显,且缺乏可靠的早期诊断手段,约有70%的患者在确诊时已进入到疾病进展期。卵巢癌的早期诊断,是关系到病人预后的最重要的因素。基因芯片技术已成为卵巢癌的早期诊断方法之一。外周血单个核细胞(peripheral blood mononuclear cells,PBMC)除淋巴细胞和单核细胞外,也包含一些循环肿瘤细胞(circulating tumor cells,CTCs)和肿瘤干细胞(tumor stem cells,TSCs),在肿瘤病患中的改变可以体现机体的微环境变化。本文利用基因芯片技术检测卵巢癌患者和健康人对照的外周血中,PBMC基因表达谱的差异,不仅有利于卵巢癌的早期诊断,且还可能揭示卵巢癌发病、转移中的机制。单链抗体(single-chain fragment variable,sc Fv)是由抗体的重链和轻链的可变区经过一段15~20个氨基酸的片段连接而成,是有功能抗体的最小的形式。单链抗体的相对分子质量小,组织穿透能力强,免疫原性低,在肿瘤的诊断和治疗上有着广泛的应用,如将单链抗体连接放射性标记物,应用于肿瘤组织的原位成像,或连接抗肿瘤的细胞毒素或化疗药物,进行肿瘤的治疗等。本文利用酵母展示技术,成功构建了卵巢癌患者的单链抗体库,并用卵巢癌A2780细胞株对抗体库进行了初步筛选,为今后进一步获取可以应用于临床的单链抗体提供了基础。目的:一、应用基因芯片技术检测卵巢癌患者和健康人对照的外周血中,PBMC的基因表达谱差异,筛选卵巢癌患者差异表达基因(differentially expressed genes,DEG),对差异基因进行基因通路(pathway)分析、基因本体论(GO)分析和功能分析。二、构建卵巢癌患者的酵母展示单链抗体库,对构建的单链抗体库测定库容进行初步评价,并用卵巢癌细胞株A2780对抗体库进行初步筛选,为进一步获取高亲和力的卵巢癌单链抗体提供基础。方法:一、提取临床确诊为卵巢癌的8例患者和4例健康女性的外周血中的PBMC样本,提取PBMC中的总RNA,将卵巢癌患者的样本两两混合成四份样本,以减少样本间差异。利用商业化基因芯片Affymetrix?Human Genome U219 Array Strip对两组样本进行转录本基因表达量的检测,利用软件和在线网站对结果进行分析。二、取13例卵巢癌患者外周血分离PBMC,TRIzol法提取PBMC总RNA,逆转录PCR合成出cDNA,利用设计的引物扩增出全套轻链可变区(VL)和重链可变区(VH)基因片段。使用重叠延伸PCR(SOE-PCR)将轻、重链基因片段连接。利用真核细胞体内同源重组系统介导的间隙修复(gap-repair)机制,将连接好的单链抗体片段和线性化的质粒p YD1同时转入酵母菌中。使用不含色氨酸的筛选培养基进行转化后的筛选。在半乳糖存在的诱导培养基中,酵母将单链抗体表达于酵母细胞表面,建立卵巢癌患者的单链抗体库。而后使用卵巢癌细胞株A2780与表达单链抗体的酵母细胞进行结合,利用流式细胞术对可结合A2780细胞的酵母菌株进行初步筛选。结果:一、基因芯片检测后分析发现,卵巢癌患者PBMC相比健康人PBMC中差异表达基因共1158个(Fold Change绝对值2倍,p0.05),其中上调的差异表达基因有311个,下调的差异表达基因847个。上调差异最大的5个基因是THBS1、NR4A2、BTG1、ADM、MIR22,下调差异最大的5个基因是GIMAP8、CX3CR1、GIMAP4、ST8SIA4、MYOM2。对差异基因进行通路分析后发现,涉及的通路主要分布在信号转导系统和免疫系统。对差异基因进行GO功能富集分析后发现,差异基因在细胞组成分析中,主要涉及细胞组分和细胞器,在分子功能分析中主要具有结合和催化活性,同时在生物学过程分析中,主要对细胞过程和代谢过程起作用。对差异基因进行功能分析后,在分类为凋亡的基因与GO分析为免疫系统过程的结果取交集,共筛选出有意义的差异基因6个。二、利用PCR方法成功扩增出部分VH、VL基因片段,VH、VL连接后和线性化的质粒共同转化进入酵母菌中,在筛选培养基中有菌落生长,表明转化成功。对建立的单链抗体库进行了库容的检测,测定库容为:κ链抗体库库容为4×109、λ链抗体库库容为8×109。使用卵巢癌细胞系A2780对抗体库进行了筛选,共得到展示VH-VLκ链阳性的酵母菌株49株,展示VH-VLλ链阳性酵母菌株90株。结论:一、卵巢癌患者和健康对照人群的PBMC中基因表达谱存在显著差异,筛选的卵巢癌患者差异基因为进一步研究卵巢癌提供了基础。二、利用酵母表面展示系统成功构建出卵巢癌的单链抗体库,并在诱导下在酵母细胞表面表达抗卵巢癌细胞的单链抗体,利用卵巢癌肿瘤细胞系A2780对抗体库进行了初步筛选,为以后进一步获取高亲和力的卵巢癌单链抗体提供了基础。
[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.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31

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