NK细胞活化性受体NKp30的表达与功能研究
发布时间：2019-06-15 06:28
【摘要】：固有免疫(innate immunity)是生物体在物种进化过程中形成的一系列防御机制,能迅速的对侵入的病原体产生非特异性抗感染免疫应答,同时固有免疫在适应性免疫应答过程中也起着重要作用。天然杀伤细胞(natural killer cells, NK)是固有免疫系统的重要组成部分,在机体防御病原微生物感染和抗肿瘤方面发挥着重要的作用。NK细胞的活化受其表面活化性与抑制性受体的协同调控,自然细胞毒性受体(natural cytotoxicity receptors, NCRs)是NK细胞特有的活化性受体,通常在NK细胞表面抑制性受体丧失识别“自我”能力时,发挥杀伤作用。 NKp30(NCR3)是NCR家族的一个重要成员,表达于所有NK细胞表面,在NK细胞活化以及杀伤肿瘤的过程中发挥重要作用,近几年研究表明,NKp30分子参与NK细胞与树突状细胞(dendritic cells, DC)的交互调节(crosstalk),实现对固有免疫和适应性免疫的调控。NKp30分子的功能在过去10年里得到了研究者广泛的关注,但NKp30分子在NK细胞的活化及免疫突触形成中的确切作用机制尚不清楚。本研究中,我们构建了人NKp30胞外段结构域原核细胞表达载体,获得有生物学活性的NKp30重组蛋白。我们采用NKp30重组蛋白免疫小鼠,制备了抗NKp30的单克隆抗体。我们利用免疫荧光、51Cr同位素释放实验、流式细胞术(FACS)和免疫印迹(Western blotting)实验对NKp30分子在NK细胞活化及NK细胞免疫突触形成中的作用进行了研究。 主要取得了以下结果: 1,NKp30分子胞外段结构域蛋白的表达与纯化 构建NKp30胞外段结构域蛋白的原核细胞重组表达载体,通过诱导表达,重组蛋白以包涵体形式存在,经过Western blotting和质谱鉴定,目的蛋白为人NKp30重组蛋白(简称rhNKp30)。分别采用稀释复性法和镍柱亲和层析法对rhNKp30进行复性和纯化,蛋白纯度可以达到95%以上。经FACS检测rhNKp30能够与NKp30配体阳性细胞结合,51Cr释放实验结果显示rhNKp30能够抑制NK92细胞对Hela细胞的杀伤。结果提示rhNKp30具有生物学活性,可用于功能研究。 2,抗人NKp30单克隆抗体的制备、鉴定及应用 用rhNKp30免疫8-10周龄的BALB/c雌性小鼠,采取不同的免疫策略,经过5次免疫后,取小鼠的脾脏细胞与骨髓瘤SP2/0细胞在聚乙二醇作用下进行体外融合。通过有限稀释法和间接酶联免疫吸附试验(ELISA)筛选阳性杂交瘤细胞株,共获得了4株能稳定分泌抗rhNKp30的单克隆抗体杂交瘤细胞株。我们选择了其中对NKp30亲和力高的克隆号为3G5的单克隆抗体进行了深入研究。常规方法制备腹水,经ProteinG亲和层析法纯化获得纯度为95%的单克隆抗体3G5,并对该抗体进行了特性鉴定和应用检测。单克隆抗体3G5能广泛用于Western blotting、免疫沉淀(Immunoprecipitation)、ELISA和FACS检测实验,并且单克隆抗体3G5不影响NKp30受体与其配体的结合。 3,NKp30分子参与NK细胞活化及免疫突触形成 通过51Cr同位素释放实验检测NK细胞对靶细胞的杀伤,实验结果表明抗NKp30阻断抗体(克隆号:P30-15)能够明显抑制外周血NK细胞或者NK细胞系NK92细胞对多种靶细胞的杀伤。通过筛选发现NKp30分子在外周血NK细胞或者NK92细胞杀伤Hela细胞中发挥着主导作用。为此我们采用NK92细胞作为效应细胞,Hela细胞作为靶细胞进行免疫突触的研究。采用不同荧光标记的抗体的Confocal检测结果显示:NKp30与CD11a分子共定位而且聚集在NK92细胞与Hela细胞的接触面上,提示NKp30分子参与NK细胞免疫突触的形成。通过FACS和免疫荧光检测NK92细胞与Hela细胞的结合显示,在0-15 min内,NK92细胞和Hela细胞的结合率与孵育时间成正相关,在15 min达到最大结合率,15-30 min效靶细胞结合率变化不大。抗NKp30阻断抗体不能抑制效靶细胞的结合,提示NKp30分子不影响NK细胞与靶细胞结合。FACS检测发现,Hela细胞能刺激NK92细胞细胞毒脱颗粒的产生,抗NKp30阻断抗体能显著抑制该效应,提示NKp30分子在NK细胞脱颗粒效应中可能发挥着重要的作用。ELISA检测Hela细胞能够刺激NK92细胞分泌IFN-γ,抗NKp30阻断抗体能明显抑制NK92细胞分泌IFN-γ的水平,说明NKp30分子在NK细胞分泌细胞因子方面起着重要作用。通过Western blotting和51Cr同位素释放实验,发现PI3K-Erk1/2信号通路参与NKp30分子介导的NK细胞活化。 综上所述:NKp30分子参与NK细胞活化性免疫突触的形成,但NKp30分子不影响NK细胞与靶细胞的粘附与结合。NKp30分子识别靶细胞上的配体向NK细胞胞内传递活化信号,激活PI3K-Erk1/2信号通路,进而诱导NK细胞的脱颗粒效应与细胞因子的分泌,导致NK细胞对靶细胞的杀伤。
[Abstract]:Innate immunity is a series of defense mechanisms that the organism forms during the evolution of the species, can rapidly generate the non-specific anti-infective immune response to the invading pathogen, and the innate immunity plays an important role in the process of adaptive immune response. Natural killer cells (NK) are an important part of the innate immune system. The activation of NK cells is controlled by the co-regulation of its surface activation and inhibitory receptors, and the natural cytotoxic receptors (NCRs) are the specific activation receptors of NK cells, which usually play an anti-killing role when the NK cell surface inhibitory receptor loses its ability to recognize the "self". NKp30 (NCR3) is an important member of the NCR family, which is expressed on the surface of all NK cells and plays an important role in the activation of NK cells and the killing of tumor. In recent years, it has shown that NKp30 is involved in the interaction regulation of NK cells and dendritic cells (DC). and the modulation of the innate immunity and the adaptive immunity can be realized, The function of NKp30 molecule has been widely concerned by the researchers in the last 10 years, but the exact mechanism of NKp30 in the activation of NK cells and the formation of immune synapses is unclear. In this study, we constructed the prokaryotic cell expression vector of human NKp30 extracellular domain, and obtained the biological activity of NKp30 recombinant egg. White. We used NKp30 recombinant protein to immunize mice and prepared a monoclonal anti-NKp30 monoclonal antibody. The effects of NKp30 on the activation of NK cells and the formation of the immune synapses of NK cells were studied by means of immunofluorescence, 51Cr isotope release experiments, flow cytometry (FACS) and Western blotting. To look into. The results are as follows:1, the extracellular domain of NKp30 The prokaryotic cell recombinant expression vector of the NKp30 extracellular domain protein is constructed by the expression and purification of the expression and purification, the expression is induced, the recombinant protein is present in the form of an inclusion body, and the expression and purification of the recombinant protein are in the form of an inclusion body, ng and mass spectrometry, and the target protein is human NKp30 recombinant protein (short) HNKp30). The renaturation and purification of rhNKp30 were carried out by the dilution and renaturation method and the nickel-column affinity chromatography, respectively. By FACS, rhNKp30 can be combined with NKp30 ligand-positive cells, and the results of 51Cr release show that rhNKp30 can inhibit the inhibition of NK92 cell to He. the results suggest that rhNKp30 has biological activity, can be used for functional study.2. Anti-human NKp30 monoclonal antibody The preparation, identification and application of rhNKp30 were used to immunize 8-10-week-old BALB/ c female mice. In vitro fusion was carried out under the action of glycol. The positive hybridoma cell line was screened by a limited dilution method and an indirect enzyme-linked immunosorbent assay (ELISA), and 4 strains of anti-rhNKp30 were obtained stably. Monoclonal antibody hybridoma cell line. We selected a single clone number of 3 G5 with a high affinity for NKp30. The monoclonal antibody 3G5 with the purity of 95% was purified by the ProteinG affinity chromatography and the antibody was purified by the ProteinG affinity chromatography. The monoclonal antibody 3G5 can be widely used in Western blotting, immunoprecipitation, ELISA and FACS detection, and the monoclonal antibody 3G5 does not affect NKp. Binding of 30 receptors to their ligands. NK cell activation and the formation of immune synapses through 51Cr isotope release assay to detect the killing of NK cells on target cells, and experimental results The anti-NKp30 blocking antibody (clone number: P30-15) can obviously inhibit the peripheral blood NK cell or the NK cell line. The killing of a plurality of target cells by NK92 cells. NKp30 molecules were found to be killed in peripheral blood NK cells or NK92 cells by screening. Hela cells play a leading role in the treatment of Hela cells. We use NK92 cells as effector cells, Hela fine. The results showed that NKp30 was co-located with CD11a and was collected on the contact surface of NK92 and Hela cells. The binding rate of NK92 cells and Hela cells was positively correlated with the incubation time within 0-15 min, and the maximum binding rate was reached at 15 min,15-3. The binding rate of anti-NKp30-blocking antibody could not inhibit the binding of the target target cells, and it was suggested that NKp30 could not inhibit the binding of the target target cells. The binding of NK cells with target cells was not affected by the molecule. The results of FACS showed that the production of the cytotoxic departicles of NK92 cells could be stimulated by the Hela cells, and the anti-NKp30 blocking antibody could significantly inhibit the effect. It is possible to play an important role in the particle effect. The ELISA for the detection of Hela cells can stimulate the secretion of IFN-1 in NK92 cells, and the anti-NKp30 blocking antibody can obviously inhibit the level of the IFN-1 secreted by the NK92 cells, which indicates that the NKp30 molecule is fine in NK. The role of PI3K-Erk1/2 signaling pathway was found by Western blotting and 51Cr isotope release experiments. In conclusion, NKp30 molecules are involved in the formation of NK cell-activated immune synapses, but NKp30 molecules It does not affect the adhesion and binding of NK cells to target cells. NKp30 molecules recognize the ligand on the target cell to transfer the activation signal to the NK cell, activate the PI3K-Erk1/2 signaling pathway, and further induce the departicle effect and the cytokine of the NK cell.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392
本文编号：2500035
[Abstract]:Innate immunity is a series of defense mechanisms that the organism forms during the evolution of the species, can rapidly generate the non-specific anti-infective immune response to the invading pathogen, and the innate immunity plays an important role in the process of adaptive immune response. Natural killer cells (NK) are an important part of the innate immune system. The activation of NK cells is controlled by the co-regulation of its surface activation and inhibitory receptors, and the natural cytotoxic receptors (NCRs) are the specific activation receptors of NK cells, which usually play an anti-killing role when the NK cell surface inhibitory receptor loses its ability to recognize the "self". NKp30 (NCR3) is an important member of the NCR family, which is expressed on the surface of all NK cells and plays an important role in the activation of NK cells and the killing of tumor. In recent years, it has shown that NKp30 is involved in the interaction regulation of NK cells and dendritic cells (DC). and the modulation of the innate immunity and the adaptive immunity can be realized, The function of NKp30 molecule has been widely concerned by the researchers in the last 10 years, but the exact mechanism of NKp30 in the activation of NK cells and the formation of immune synapses is unclear. In this study, we constructed the prokaryotic cell expression vector of human NKp30 extracellular domain, and obtained the biological activity of NKp30 recombinant egg. White. We used NKp30 recombinant protein to immunize mice and prepared a monoclonal anti-NKp30 monoclonal antibody. The effects of NKp30 on the activation of NK cells and the formation of the immune synapses of NK cells were studied by means of immunofluorescence, 51Cr isotope release experiments, flow cytometry (FACS) and Western blotting. To look into. The results are as follows:1, the extracellular domain of NKp30 The prokaryotic cell recombinant expression vector of the NKp30 extracellular domain protein is constructed by the expression and purification of the expression and purification, the expression is induced, the recombinant protein is present in the form of an inclusion body, and the expression and purification of the recombinant protein are in the form of an inclusion body, ng and mass spectrometry, and the target protein is human NKp30 recombinant protein (short) HNKp30). The renaturation and purification of rhNKp30 were carried out by the dilution and renaturation method and the nickel-column affinity chromatography, respectively. By FACS, rhNKp30 can be combined with NKp30 ligand-positive cells, and the results of 51Cr release show that rhNKp30 can inhibit the inhibition of NK92 cell to He. the results suggest that rhNKp30 has biological activity, can be used for functional study.2. Anti-human NKp30 monoclonal antibody The preparation, identification and application of rhNKp30 were used to immunize 8-10-week-old BALB/ c female mice. In vitro fusion was carried out under the action of glycol. The positive hybridoma cell line was screened by a limited dilution method and an indirect enzyme-linked immunosorbent assay (ELISA), and 4 strains of anti-rhNKp30 were obtained stably. Monoclonal antibody hybridoma cell line. We selected a single clone number of 3 G5 with a high affinity for NKp30. The monoclonal antibody 3G5 with the purity of 95% was purified by the ProteinG affinity chromatography and the antibody was purified by the ProteinG affinity chromatography. The monoclonal antibody 3G5 can be widely used in Western blotting, immunoprecipitation, ELISA and FACS detection, and the monoclonal antibody 3G5 does not affect NKp. Binding of 30 receptors to their ligands. NK cell activation and the formation of immune synapses through 51Cr isotope release assay to detect the killing of NK cells on target cells, and experimental results The anti-NKp30 blocking antibody (clone number: P30-15) can obviously inhibit the peripheral blood NK cell or the NK cell line. The killing of a plurality of target cells by NK92 cells. NKp30 molecules were found to be killed in peripheral blood NK cells or NK92 cells by screening. Hela cells play a leading role in the treatment of Hela cells. We use NK92 cells as effector cells, Hela fine. The results showed that NKp30 was co-located with CD11a and was collected on the contact surface of NK92 and Hela cells. The binding rate of NK92 cells and Hela cells was positively correlated with the incubation time within 0-15 min, and the maximum binding rate was reached at 15 min,15-3. The binding rate of anti-NKp30-blocking antibody could not inhibit the binding of the target target cells, and it was suggested that NKp30 could not inhibit the binding of the target target cells. The binding of NK cells with target cells was not affected by the molecule. The results of FACS showed that the production of the cytotoxic departicles of NK92 cells could be stimulated by the Hela cells, and the anti-NKp30 blocking antibody could significantly inhibit the effect. It is possible to play an important role in the particle effect. The ELISA for the detection of Hela cells can stimulate the secretion of IFN-1 in NK92 cells, and the anti-NKp30 blocking antibody can obviously inhibit the level of the IFN-1 secreted by the NK92 cells, which indicates that the NKp30 molecule is fine in NK. The role of PI3K-Erk1/2 signaling pathway was found by Western blotting and 51Cr isotope release experiments. In conclusion, NKp30 molecules are involved in the formation of NK cell-activated immune synapses, but NKp30 molecules It does not affect the adhesion and binding of NK cells to target cells. NKp30 molecules recognize the ligand on the target cell to transfer the activation signal to the NK cell, activate the PI3K-Erk1/2 signaling pathway, and further induce the departicle effect and the cytokine of the NK cell.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 刘映霞,胡国龄,刘敏,何淑雅,谭德明,李红梅;抗NKG2D多克隆抗体抑制NK和LAK细胞细胞毒效应的研究[J];细胞与分子免疫学杂志;2003年03期
，本文编号：2500035
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