EBV体外转染人B淋巴细胞方法的优化及B淋巴母细胞样细胞系的体外建立

发布时间：2018-06-25 00:49

本文选题：EB病毒 + CpG　；参考：《苏州大学》2009年硕士论文

【摘要】： B淋巴细胞是机体体液免疫的主要效应细胞,以表达和分泌免疫球蛋白为特征。从某种意义上讲,机体的体液免疫应答过程就是B细胞针对特异抗原而活化、增殖和分化过程。检测B细胞特性及免疫球蛋白,了解机体体液免疫状况,对于探讨各种疾病的免疫发病机理及药物治疗效应均有重要的临床意义;分析B细胞发育不同阶段的表面标记,了解B细胞发育和分化阶段的特性与疾病的关系,可为临床提供诊治相关疾病的有用资料。 EB病毒(Epstein-Barr Virus,EBV)是一种γ亚科疱疹病毒,它与人类很多恶性疾病有关,特别是上皮和淋巴起源的肿瘤。EBV在体外能感染正常静止的B细胞,使它们变成永生化的淋巴细胞系(LCL)。由于EBV的这一特性,目前已被广泛应用于多种细胞的永生化,转化的细胞株染色体稳定,保留了原有的遗传性状。病毒转化后的B淋巴母细胞样细胞系(B-LCLs)依然保存了B细胞的分化特征和免疫表型,作为抗原提呈细胞,它可以摄取加工抗原,并将其载体部分表达于细胞表面,供T细胞受体识别;也可作为抗体基因的来源,将其与骨髓瘤细胞融合,制备分泌相应的单克隆抗体的杂交瘤细胞株。近年尤其值得关注的是,利用B-LCLs生物学特性作为人源化抗体的生产体系。 CpG基序(motif)可以活化免疫细胞,激发机体产生Th1型为主的免疫应答。目前的研究已证明:CpG寡核苷酸能够活化人类B淋巴细胞,诱导多种细胞因子和细胞表面免疫相关分子的表达。B细胞上高表达重要的免疫分子CD40,CD40mAb与B细胞上的CD40分子发生交联,而使B细胞免于凋亡和增加生存能力。因此本实验旨在探讨CpG基序和CD40激发型抗体(5C11)对EBV转化的作用,从而优化B-LCLs建系的方法,对于体外利用和研究B淋巴细胞株具有重要的理论价值和潜在的运用意义。第一部分EBV体外转染人B细胞方法的优化目的探讨体外建立健康人外周血的B淋巴母细胞样细胞系(LCLs)的优化方法及其影响因素。方法用EB病毒感染健康人外周血中分离的单个核细胞(PBMC),加入CD40激发型抗体(5C11)、CpG DNA免疫调节基序以诱导B淋巴细胞增殖,环胞菌素A(CysA)抑制T淋巴细胞的活性。光学显微镜下观察LCLs的形态特征,利用流式细胞术分析LCLs膜表面分子CD19的表达水平。结果PBMC经EBV感染3周后转化成永生化B淋巴母细胞系。转化后的B淋巴母细胞体积增大积聚成团,可进一步分裂增殖并长期传代培养。CD40激发型抗体(5C11)及CpG免疫调节基序联合运用,明显地提高了EBV转化效率,转化后的LCLs保持了成熟B淋巴细胞的生物学特征。结论CD40信号激发和CpG免疫调节基序联合运用有效地促进EBV对人B淋巴细胞的转化及体外建系。第二部分人B淋巴母细胞系的体外建立及生物学活性鉴定目的体外建立健康人外周血的B淋巴母细胞样细胞系(LCLs)并鉴定其生物学活性。方法光学显微镜下观察,并做Giemsa染色,观察LCLs的形态特征;提取LCLs的总RNA,检测EB病毒潜伏膜蛋白基因LMP1的存在;利用流式细胞术分析LCLs膜表面分子CD19、CD40的表达水平。结果PBMC经EBV感染3周后转化成永生化B淋巴母细胞系。转化后的B淋巴母细胞体积增大积聚成团,可进一步分裂增殖并长期传代培养。转化后的LCLs含有LMP1基因,并保持了成熟B淋巴细胞的生物学特征。结论用EBV体外转染方法成功建立B淋巴母细胞样细胞系,转化后的B淋巴母细胞能持续分裂,并保持了成熟B淋巴细胞的生物学特性。
[Abstract]:B lymphocyte is the main effect cell of body humoral immunity, which is characterized by expression and secretion of immunoglobulin. In a sense, the body humoral immune response process is the process of activation, proliferation and differentiation of B cells against specific antigens. Detection of the characteristics of B cells and immunoglobulin, understanding the body humoral immune status, and the study of the body fluid immunity The immune mechanism of various diseases and the effect of drug treatment have important clinical significance. The analysis of the surface markers at different stages of B cell development and the understanding of the relationship between the characteristics of the development and differentiation of B cells and the disease can provide useful information for the clinical diagnosis and treatment of related diseases.
EB virus (Epstein-Barr Virus, EBV) is a kind of gamma subfamily herpes virus, which is associated with many human malignant diseases, especially the epithelial and lymphoid cancer.EBV can infect normal stationary B cells in vitro, and make them become immortalized lymphocyte lines (LCL). Because of this characteristic of EBV, it is now widely used in many kinds of cells. Immortalized, the transformed cell line has a stable chromosome and retained the original genetic character. The transformed B lymphoblastic cell line (B-LCLs) still preserves the differentiation and immunophenotype of the B cells. As an antigen presenting cell, it can absorb the processed antigen and express its carrier part on the cell surface for the T cell receptor recognition. It can also be used as a source of antibody gene and the fusion of myeloma cells to produce hybridoma cell lines that secrete the corresponding monoclonal antibodies. In recent years, it is particularly noteworthy that the biological characteristics of B-LCLs are used as a production system for humanized antibodies.
CpG motif (motif) can activate immune cells and stimulate the organism to produce Th1 based immune responses. Current studies have shown that CpG oligonucleotides can activate human B lymphocytes and induce a variety of cytokines and cell surface immuno related molecules to express the important immune molecule CD40 on.B cells, CD40mAb and CD40 on B cells. In this experiment, the purpose of this experiment is to explore the effect of CpG motif and CD40 excited antibody (5C11) on the transformation of EBV and to optimize the method of establishing the B-LCLs system, which has important theoretical value and potential application significance for the use and study of the B lymphocyte strain in vitro, so that the B cells are free of apoptosis and increase the survival ability.
Part one optimization of EBV transfection of human B cells in vitro
Objective to explore the optimization of B lymphoblastic cell line (LCLs) of healthy human peripheral blood in vitro and its influencing factors. Methods using EB virus to infect mononuclear cells (PBMC) isolated from peripheral blood of healthy people, CD40 excited antibody (5C11), CpG DNA immunomodulatory sequence to induce B lymphocyte proliferation and cyclosporin A (CysA) inhibition T lymphocyte activity. The morphological characteristics of LCLs were observed under the optical microscope. The expression level of the molecular CD19 on the surface of LCLs membrane was analyzed by flow cytometry. Results PBMC was transformed into immortalized B lymphoblastic line after 3 weeks of EBV infection. The transformed B lymphoblastoid cell volume increased and formed into a group, which could further split and proliferate and culture for a long time. The combined use of CD40 activated antibody (5C11) and CpG immunomodulatory sequence significantly improved the efficiency of EBV transformation. The transformed LCLs maintained the biological characteristics of the mature B lymphocyte. Conclusion the combined use of CD40 signal and CpG immunomodulatory motif can effectively promote the transformation of EBV to human B lymphoblastic cells and its construction in vitro.
Establishment and biological activity identification of second human B lymphoblastoid cell lines in vitro
Objective to establish the B lymphoblastic cell line (LCLs) of healthy human peripheral blood in vitro and to identify its biological activity. Methods the morphological characteristics of LCLs were observed by optical microscope and Giemsa staining was done. The total RNA of LCLs was extracted and the survival of the latent membrane protein gene LMP1 of EB virus was detected, and the molecular CD19 of LCLs membrane surface molecule CD19 and CD were analyzed by flow cytometry. 40 of the expression level. Results PBMC was transformed into immortalized B lymphoblastic line after 3 weeks of EBV infection. The transformed B lymphoblastoid cell volume increased and formed into a group, which could further split and proliferate and long passages. The transformed LCLs contained the LMP1 gene and maintained the biological characteristics of the mature B lymphocyte. Conclusion the transfection method of EBV in vitro was used. The B lymphoblastoid cell line was successfully established, and the transformed B lymphoblastoid cells continued to divide, and maintained the biological characteristics of mature B lymphocytes.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【引证文献】