本文選題：EB病毒 + CpG　； 參考：《蘇州大學(xué)》2009年碩士論文

【摘要】： B淋巴細(xì)胞是機(jī)體體液免疫的主要效應(yīng)細(xì)胞,以表達(dá)和分泌免疫球蛋白為特征。從某種意義上講,機(jī)體的體液免疫應(yīng)答過程就是B細(xì)胞針對(duì)特異抗原而活化、增殖和分化過程。檢測(cè)B細(xì)胞特性及免疫球蛋白,了解機(jī)體體液免疫狀況,對(duì)于探討各種疾病的免疫發(fā)病機(jī)理及藥物治療效應(yīng)均有重要的臨床意義;分析B細(xì)胞發(fā)育不同階段的表面標(biāo)記,了解B細(xì)胞發(fā)育和分化階段的特性與疾病的關(guān)系,可為臨床提供診治相關(guān)疾病的有用資料。 EB病毒(Epstein-Barr Virus,EBV)是一種γ亞科皰疹病毒,它與人類很多惡性疾病有關(guān),特別是上皮和淋巴起源的腫瘤。EBV在體外能感染正常靜止的B細(xì)胞,使它們變成永生化的淋巴細(xì)胞系(LCL)。由于EBV的這一特性,目前已被廣泛應(yīng)用于多種細(xì)胞的永生化,轉(zhuǎn)化的細(xì)胞株染色體穩(wěn)定,保留了原有的遺傳性狀。病毒轉(zhuǎn)化后的B淋巴母細(xì)胞樣細(xì)胞系(B-LCLs)依然保存了B細(xì)胞的分化特征和免疫表型,作為抗原提呈細(xì)胞,它可以攝取加工抗原,并將其載體部分表達(dá)于細(xì)胞表面,供T細(xì)胞受體識(shí)別;也可作為抗體基因的來源,將其與骨髓瘤細(xì)胞融合,制備分泌相應(yīng)的單克隆抗體的雜交瘤細(xì)胞株。近年尤其值得關(guān)注的是,利用B-LCLs生物學(xué)特性作為人源化抗體的生產(chǎn)體系。 CpG基序(motif)可以活化免疫細(xì)胞,激發(fā)機(jī)體產(chǎn)生Th1型為主的免疫應(yīng)答。目前的研究已證明:CpG寡核苷酸能夠活化人類B淋巴細(xì)胞,誘導(dǎo)多種細(xì)胞因子和細(xì)胞表面免疫相關(guān)分子的表達(dá)。B細(xì)胞上高表達(dá)重要的免疫分子CD40,CD40mAb與B細(xì)胞上的CD40分子發(fā)生交聯(lián),而使B細(xì)胞免于凋亡和增加生存能力。因此本實(shí)驗(yàn)旨在探討CpG基序和CD40激發(fā)型抗體(5C11)對(duì)EBV轉(zhuǎn)化的作用,從而優(yōu)化B-LCLs建系的方法,對(duì)于體外利用和研究B淋巴細(xì)胞株具有重要的理論價(jià)值和潛在的運(yùn)用意義。 第一部分EBV體外轉(zhuǎn)染人B細(xì)胞方法的優(yōu)化 目的探討體外建立健康人外周血的B淋巴母細(xì)胞樣細(xì)胞系(LCLs)的優(yōu)化方法及其影響因素。方法用EB病毒感染健康人外周血中分離的單個(gè)核細(xì)胞(PBMC),加入CD40激發(fā)型抗體(5C11)、CpG DNA免疫調(diào)節(jié)基序以誘導(dǎo)B淋巴細(xì)胞增殖,環(huán)胞菌素A(CysA)抑制T淋巴細(xì)胞的活性。光學(xué)顯微鏡下觀察LCLs的形態(tài)特征,利用流式細(xì)胞術(shù)分析LCLs膜表面分子CD19的表達(dá)水平。結(jié)果PBMC經(jīng)EBV感染3周后轉(zhuǎn)化成永生化B淋巴母細(xì)胞系。轉(zhuǎn)化后的B淋巴母細(xì)胞體積增大積聚成團(tuán),可進(jìn)一步分裂增殖并長(zhǎng)期傳代培養(yǎng)。CD40激發(fā)型抗體(5C11)及CpG免疫調(diào)節(jié)基序聯(lián)合運(yùn)用,明顯地提高了EBV轉(zhuǎn)化效率,轉(zhuǎn)化后的LCLs保持了成熟B淋巴細(xì)胞的生物學(xué)特征。結(jié)論CD40信號(hào)激發(fā)和CpG免疫調(diào)節(jié)基序聯(lián)合運(yùn)用有效地促進(jìn)EBV對(duì)人B淋巴細(xì)胞的轉(zhuǎn)化及體外建系。 第二部分人B淋巴母細(xì)胞系的體外建立及生物學(xué)活性鑒定 目的體外建立健康人外周血的B淋巴母細(xì)胞樣細(xì)胞系(LCLs)并鑒定其生物學(xué)活性。方法光學(xué)顯微鏡下觀察,并做Giemsa染色,觀察LCLs的形態(tài)特征;提取LCLs的總RNA,檢測(cè)EB病毒潛伏膜蛋白基因LMP1的存在;利用流式細(xì)胞術(shù)分析LCLs膜表面分子CD19、CD40的表達(dá)水平。結(jié)果PBMC經(jīng)EBV感染3周后轉(zhuǎn)化成永生化B淋巴母細(xì)胞系。轉(zhuǎn)化后的B淋巴母細(xì)胞體積增大積聚成團(tuán),可進(jìn)一步分裂增殖并長(zhǎng)期傳代培養(yǎng)。轉(zhuǎn)化后的LCLs含有LMP1基因,并保持了成熟B淋巴細(xì)胞的生物學(xué)特征。結(jié)論用EBV體外轉(zhuǎn)染方法成功建立B淋巴母細(xì)胞樣細(xì)胞系,轉(zhuǎn)化后的B淋巴母細(xì)胞能持續(xù)分裂,并保持了成熟B淋巴細(xì)胞的生物學(xué)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉宇;趙玉華;;細(xì)胞永生化技術(shù)在體育領(lǐng)域研究的可行性分析[J];哈爾濱體育學(xué)院學(xué)報(bào);2011年01期

相關(guān)碩士學(xué)位論文 前1條

1 劉宇;我國(guó)優(yōu)秀單板U型場(chǎng)地滑雪運(yùn)動(dòng)員永生細(xì)胞庫(kù)的建立與保存[D];哈爾濱體育學(xué)院;2011年

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本文編號(hào)：2063742