NK細胞活化性受體NKp30的表達與功能研究
發(fā)布時間：2019-06-15 06:28
【摘要】：固有免疫(innate immunity)是生物體在物種進化過程中形成的一系列防御機制,能迅速的對侵入的病原體產(chǎn)生非特異性抗感染免疫應答,同時固有免疫在適應性免疫應答過程中也起著重要作用。天然殺傷細胞(natural killer cells, NK)是固有免疫系統(tǒng)的重要組成部分,在機體防御病原微生物感染和抗腫瘤方面發(fā)揮著重要的作用。NK細胞的活化受其表面活化性與抑制性受體的協(xié)同調(diào)控,自然細胞毒性受體(natural cytotoxicity receptors, NCRs)是NK細胞特有的活化性受體,通常在NK細胞表面抑制性受體喪失識別“自我”能力時,發(fā)揮殺傷作用。 NKp30(NCR3)是NCR家族的一個重要成員,表達于所有NK細胞表面,在NK細胞活化以及殺傷腫瘤的過程中發(fā)揮重要作用,近幾年研究表明,NKp30分子參與NK細胞與樹突狀細胞(dendritic cells, DC)的交互調(diào)節(jié)(crosstalk),實現(xiàn)對固有免疫和適應性免疫的調(diào)控。NKp30分子的功能在過去10年里得到了研究者廣泛的關(guān)注,但NKp30分子在NK細胞的活化及免疫突觸形成中的確切作用機制尚不清楚。本研究中,我們構(gòu)建了人NKp30胞外段結(jié)構(gòu)域原核細胞表達載體,獲得有生物學活性的NKp30重組蛋白。我們采用NKp30重組蛋白免疫小鼠,制備了抗NKp30的單克隆抗體。我們利用免疫熒光、51Cr同位素釋放實驗、流式細胞術(shù)(FACS)和免疫印跡(Western blotting)實驗對NKp30分子在NK細胞活化及NK細胞免疫突觸形成中的作用進行了研究。 主要取得了以下結(jié)果: 1,NKp30分子胞外段結(jié)構(gòu)域蛋白的表達與純化 構(gòu)建NKp30胞外段結(jié)構(gòu)域蛋白的原核細胞重組表達載體,通過誘導表達,重組蛋白以包涵體形式存在,經(jīng)過Western blotting和質(zhì)譜鑒定,目的蛋白為人NKp30重組蛋白(簡稱rhNKp30)。分別采用稀釋復性法和鎳柱親和層析法對rhNKp30進行復性和純化,蛋白純度可以達到95%以上。經(jīng)FACS檢測rhNKp30能夠與NKp30配體陽性細胞結(jié)合,51Cr釋放實驗結(jié)果顯示rhNKp30能夠抑制NK92細胞對Hela細胞的殺傷。結(jié)果提示rhNKp30具有生物學活性,可用于功能研究。 2,抗人NKp30單克隆抗體的制備、鑒定及應用 用rhNKp30免疫8-10周齡的BALB/c雌性小鼠,采取不同的免疫策略,經(jīng)過5次免疫后,取小鼠的脾臟細胞與骨髓瘤SP2/0細胞在聚乙二醇作用下進行體外融合。通過有限稀釋法和間接酶聯(lián)免疫吸附試驗(ELISA)篩選陽性雜交瘤細胞株,共獲得了4株能穩(wěn)定分泌抗rhNKp30的單克隆抗體雜交瘤細胞株。我們選擇了其中對NKp30親和力高的克隆號為3G5的單克隆抗體進行了深入研究。常規(guī)方法制備腹水,經(jīng)ProteinG親和層析法純化獲得純度為95%的單克隆抗體3G5,并對該抗體進行了特性鑒定和應用檢測。單克隆抗體3G5能廣泛用于Western blotting、免疫沉淀(Immunoprecipitation)、ELISA和FACS檢測實驗,并且單克隆抗體3G5不影響NKp30受體與其配體的結(jié)合。 3,NKp30分子參與NK細胞活化及免疫突觸形成 通過51Cr同位素釋放實驗檢測NK細胞對靶細胞的殺傷,實驗結(jié)果表明抗NKp30阻斷抗體(克隆號:P30-15)能夠明顯抑制外周血NK細胞或者NK細胞系NK92細胞對多種靶細胞的殺傷。通過篩選發(fā)現(xiàn)NKp30分子在外周血NK細胞或者NK92細胞殺傷Hela細胞中發(fā)揮著主導作用。為此我們采用NK92細胞作為效應細胞,Hela細胞作為靶細胞進行免疫突觸的研究。采用不同熒光標記的抗體的Confocal檢測結(jié)果顯示:NKp30與CD11a分子共定位而且聚集在NK92細胞與Hela細胞的接觸面上,提示NKp30分子參與NK細胞免疫突觸的形成。通過FACS和免疫熒光檢測NK92細胞與Hela細胞的結(jié)合顯示,在0-15 min內(nèi),NK92細胞和Hela細胞的結(jié)合率與孵育時間成正相關(guān),在15 min達到最大結(jié)合率,15-30 min效靶細胞結(jié)合率變化不大�？筃Kp30阻斷抗體不能抑制效靶細胞的結(jié)合,提示NKp30分子不影響NK細胞與靶細胞結(jié)合。FACS檢測發(fā)現(xiàn),Hela細胞能刺激NK92細胞細胞毒脫顆粒的產(chǎn)生,抗NKp30阻斷抗體能顯著抑制該效應,提示NKp30分子在NK細胞脫顆粒效應中可能發(fā)揮著重要的作用。ELISA檢測Hela細胞能夠刺激NK92細胞分泌IFN-γ,抗NKp30阻斷抗體能明顯抑制NK92細胞分泌IFN-γ的水平,說明NKp30分子在NK細胞分泌細胞因子方面起著重要作用。通過Western blotting和51Cr同位素釋放實驗,發(fā)現(xiàn)PI3K-Erk1/2信號通路參與NKp30分子介導的NK細胞活化。 綜上所述:NKp30分子參與NK細胞活化性免疫突觸的形成,但NKp30分子不影響NK細胞與靶細胞的粘附與結(jié)合。NKp30分子識別靶細胞上的配體向NK細胞胞內(nèi)傳遞活化信號,激活PI3K-Erk1/2信號通路,進而誘導NK細胞的脫顆粒效應與細胞因子的分泌,導致NK細胞對靶細胞的殺傷。
[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.
【學位授予單位】：中國科學技術(shù)大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R392
本文編號：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.
【學位授予單位】：中國科學技術(shù)大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R392
【參考文獻】
相關(guān)期刊論文 前1條
1 劉映霞,胡國齡,劉敏,何淑雅,譚德明,李紅梅;抗NKG2D多克隆抗體抑制NK和LAK細胞細胞毒效應的研究[J];細胞與分子免疫學雜志;2003年03期
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