【摘要】：目的：干擾素-γ (IFN-γ)是在特定刺激作用下由細(xì)胞產(chǎn)生的一種具有高度生物活性的糖蛋白,在同種動(dòng)物細(xì)胞上具有抗腫瘤和抗病毒活性及廣泛的免疫調(diào)控作用。內(nèi)源性的IFN-γ表達(dá)水平的高低在很大程度上能反映機(jī)體的細(xì)胞免疫狀態(tài),因此IFN-γ的檢測(cè)技術(shù)在免疫學(xué)的基礎(chǔ)研究及多種病原感染的診斷中得到了廣泛的應(yīng)用。傳統(tǒng)的IFN-γ檢測(cè)方法,通常是以特異性抗IFN-γ抗體為基礎(chǔ)所建立的。雖然這種方法已相對(duì)成熟,但目前商品化的試劑盒在大規(guī)模檢測(cè)時(shí)存在成本較高的問題,因而有必要研發(fā)更為經(jīng)濟(jì)有效的檢測(cè)方法及新型的可用于IFN-γ檢測(cè)的配體。單鏈DNA或RNA可以借由自身所形成的三維空間結(jié)構(gòu)與多種靶標(biāo)如蛋白質(zhì)、多肽及其他眾多的化合物結(jié)合,這種能與靶標(biāo)以高親和力特異性結(jié)合的寡核苷酸稱為核酸適配體(aptamer)。適配體具有靶標(biāo)范圍廣,親和力高,特異性強(qiáng),易于合成價(jià)格低廉等特點(diǎn),其作用機(jī)制與單克隆抗體類似,通過氫鍵或范德華力與靶標(biāo)特異性地結(jié)合,起到識(shí)別靶標(biāo)分子或抑制其生物學(xué)活性的作用。鑒于以上特點(diǎn),適配體在腫瘤的靶向治療以及分子檢測(cè)等領(lǐng)域被廣泛應(yīng)用,因此有可能利用核酸適配體開發(fā)出更為經(jīng)濟(jì)、高效的IFN-γ檢測(cè)方法。在本研究中,我們篩選到一種全新的INF-γ核酸適配體,并建立了一種基于該適配體的IFN-γ檢測(cè)方法,用于測(cè)量淋巴細(xì)胞內(nèi)所產(chǎn)生的IFN-γ的分泌水平。 方法：我們選取正常及多聚甲醛固定過的重組人IFN-γ蛋白作為靶標(biāo),用SELEX技術(shù)篩選出了一種與該蛋白具有較高結(jié)合力的aptamer (B1-4);以流式細(xì)胞儀檢測(cè)B1-4與白蛋白、正常及固定過的IFN-γ蛋白的結(jié)合情況；抽取健康志愿者外周血,常規(guī)分離外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cells, PBMC),以phorbol12-myristate13-acetate(PMA)和Ionomycin共刺激,brefeldin A抑制蛋白轉(zhuǎn)運(yùn)：將熒光標(biāo)記的B1-4作為抗體的替代物,以流式細(xì)胞儀檢測(cè)其在刺激后的淋巴細(xì)胞內(nèi)與IFN-γ的結(jié)合情況；構(gòu)建基于適配體的胞內(nèi)IFN-γ檢測(cè)曲線。 結(jié)果：(1)我們所篩選到的IFN-γ的適配體B14,其長(zhǎng)度為59個(gè)堿基。經(jīng)測(cè)序確定,其一級(jí)序列為5'-CCGCCCAAATCCCTAAGAGAAGACTGTAATGACATCAAACCAGACACACTACACACGC A-3'。(2)該適配體與IFN-γ蛋白產(chǎn)生較強(qiáng)的結(jié)合,其親和系數(shù)為155nM。(3)該核酸適配體能夠識(shí)別未經(jīng)修飾的IFN-γ蛋白以及經(jīng)多聚甲醛固定的IFN-γ蛋白。(4)進(jìn)一步檢測(cè),該適配體與白蛋白的交叉結(jié)合相對(duì)較弱,明顯優(yōu)于國(guó)際文獻(xiàn)報(bào)道過的IFN-γ適配體。(5)該適配體能夠進(jìn)入固定后的淋巴細(xì)胞,并與經(jīng)刺激后細(xì)胞內(nèi)產(chǎn)生的IFN-γ特異性地結(jié)合。(6)隨著產(chǎn)生IFN-γ細(xì)胞比例的上升,適配體B14與細(xì)胞結(jié)合的熒光強(qiáng)度也相應(yīng)的上升,二者間呈正相關(guān)性。(7)由此我們構(gòu)建了基于核酸適配體的胞內(nèi)IFN-γ的檢測(cè)曲線,用于檢測(cè)群體淋巴細(xì)胞INF-γ的分泌水平。 結(jié)論：本研究篩選出了一種全新的IFN-γ適配體B1-4,能夠識(shí)別未修飾及經(jīng)多聚甲醛固定的IFN-γ蛋白。該適配體能夠進(jìn)入經(jīng)多聚甲醛固定的淋巴細(xì)胞,并與細(xì)胞內(nèi)的IFN-γ選擇性地結(jié)合。由此我們建立了一種新型的基于核酸適配體的胞內(nèi)IFN-γ的檢測(cè)方法。由于內(nèi)源性的IFN-γ表達(dá)水平的高低在很大程度上能反映機(jī)體的細(xì)胞免疫狀態(tài),在生物醫(yī)學(xué)研究中被廣泛檢測(cè),而適配體親和力高、特異性強(qiáng)、易于合成且價(jià)格低廉,因此我們所建立的基于核酸適配體的新型胞內(nèi)IFN-γ檢測(cè)方法在臨床檢驗(yàn)和基礎(chǔ)研究方面都具有較大的應(yīng)用潛能,為測(cè)量淋巴細(xì)胞內(nèi)干擾素的分泌提供了全新的技術(shù)手段。
[Abstract]:Objective: Interferon-1 (IFN-1) is a kind of glycoprotein with high biological activity under specific stimulation, and has anti-tumor and anti-viral activity and extensive immune regulation in the same animal cell. The level of endogenous IFN-1 expression can reflect the cellular immune status of the body to a large extent, so the detection technology of IFN-1 has been widely used in the research of immunology and the diagnosis of multiple pathogenic infections. The conventional IFN-antigen detection method is generally established on the basis of specific anti-IFN-antigen antibodies. Although the method is relatively mature, the currently commercialized kit has a high cost in large-scale detection, and therefore it is necessary to develop a more economical and effective detection method and a novel ligand which can be used for IFN-antigen detection. The single-stranded DNA or RNA can be combined with a variety of targets such as proteins, polypeptides and other numerous compounds by a three-dimensional space structure formed by itself, which is called a nucleic acid aptamer capable of specifically binding to a target with high affinity. The aptamer has the characteristics of wide target range, high affinity, strong specificity, easy synthesis and low cost, and the like, and the action mechanism is similar to the monoclonal antibody, and can be specifically combined with the target through the hydrogen bond or the Van der Waals force, so as to play the role of identifying the target molecule or inhibiting the biological activity of the target molecule. In view of the above features, the aptamer is widely used in the fields of tumor targeting therapy and molecular detection, and therefore it is possible to use the nucleic acid aptamer to develop a more economical and efficient method for detecting the IFN-antigen. In this study, we screened a brand-new type of INF-1 nucleic acid aptamer and a method of IFN-mediated detection based on the aptamer was established to measure the level of IFN-mediated secretion in lymphocytes. Methods: We selected the recombinant human IFN-2 protein fixed by normal and paraformaldehyde as the target, and the aptamer (B1-4) with a high binding force with the protein was screened by the SELEX technique, and the B1-4 and the white were detected by flow cytometry. Binding of proteins, normal and fixed IFN-I proteins; extraction of peripheral blood of healthy volunteers, conventional isolation of peripheral blood mononuclear cells (PBMCs), phorbol12-myrite13-acetate (PMA) and Ionein co-stimulation, break in A inhibition of protein transport: fluorescent labeled B1-4 as an antibody Generation of a surrogate, in the presence of a flow cytometer to detect its binding to the IFN-antigen in the stimulated lymphocytes, and the construction of an in-cell IFN-antigen assay based on the aptamer Curve. Results: (1) We screened the aptamer B14 of the IFN-1, the length of which is 59 bases. (3) the nucleic acid aptamer is capable of identifying an unmodified IFN-binding protein as well as a polyformaldehyde-immobilized IFN-binding protein. (4) further detection of the cross-junction of the aptamer with the albumin (5) the aptamer can enter the immobilized lymphocyte and specifically bind to the IFN-antigen produced in the post-stimulated cell. (6) With the generation of the IFN-antigen cell, In proportion, the fluorescence intensity of the aptamer B14 combined with the cells is also increased correspondingly, and the positive correlation is between the two. (7) The detection curve of the intracellular IFN-antigen based on the nucleic acid aptamer is thus constructed, and is used for detecting the group lymphocytes INF-1. secretion Conclusion: In this study, a new type of IFN-based aptamer B1-4 is selected, which can identify the unmodified and paraformaldehyde-immobilized IFN-I protein. The aptamer can enter the polymethylene Aldehyde-immobilized lymphocytes are selectively bound to the IFN-1 in the cell. Thus, a novel method for the detection of an intracellular IFN-antigen based on a nucleic acid aptamer is established. Since the level of endogenous IFN-antigen expression can be reflected to a large extent, The cellular immune state of the body is widely detected in the biomedical research, and the aptamer has high affinity, strong specificity, easy synthesis and low cost, so the novel intracellular IFN-antigen detection method based on the nucleic acid aptamer has the advantages of clinical examination and basic research has a large application potential and is used for measuring the secretion of the interferon in the lymphocytes,
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R392

【共引文獻(xiàn)】

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