ILT2在結(jié)核患者NK細(xì)胞的表達(dá)及作用研究
發(fā)布時(shí)間：2018-08-11 19:39
【摘要】：結(jié)核病是引起死亡人數(shù)高居第二位的感染性疾病。由于耐藥結(jié)核的產(chǎn)生、艾滋病感染流行、人口流動(dòng)和臨床免疫抑制劑的應(yīng)用增多等原因，全球結(jié)核病疫情非常嚴(yán)重，僅2012年，全球新發(fā)結(jié)核病例達(dá)860萬，有130萬人死于結(jié)核病，約45萬人為耐多藥結(jié)核病。我國現(xiàn)有活動(dòng)性肺結(jié)核病人約500萬，每年約有13萬人死于結(jié)核病。盡管結(jié)核感染人數(shù)眾多，但是僅有5-10%的感染者發(fā)展成為活動(dòng)性結(jié)核。研究結(jié)果表明，結(jié)核病的發(fā)生、發(fā)展和轉(zhuǎn)歸不僅僅取決于結(jié)核分枝桿菌的數(shù)量和毒力,在很大程度上還取決于機(jī)體的免疫狀態(tài)。 宿主固有免疫在結(jié)核病早期發(fā)揮抗結(jié)核分枝桿菌感染作用，而且對于激發(fā)宿主獲得性免疫也發(fā)揮了重要作用。在結(jié)核分枝桿菌入侵早期，獲得性免疫未被充分激活之前，固有免疫系統(tǒng)在結(jié)核分枝桿菌的控制和清除過程中發(fā)揮著重要作用。參與固有免疫細(xì)胞主要為NK細(xì)胞、巨噬細(xì)胞、單核細(xì)胞、嗜中性白細(xì)胞等，它們無須抗原預(yù)先致敏,主要通過釋放細(xì)胞因子、穿孔素、顆粒酶、表達(dá)配體等發(fā)揮作用，誘導(dǎo)免疫細(xì)胞聚集，殺傷靶細(xì)胞。 NK細(xì)胞是參與抗結(jié)核固有免疫的重要細(xì)胞，具有細(xì)胞毒性效應(yīng)，能分泌IFN-γ等細(xì)胞因子，同時(shí)具有免疫調(diào)節(jié)的功能。為了保持抗感染免疫的平衡，NK細(xì)胞的功能由位于細(xì)胞表面不同的激活和抑制型受體共同進(jìn)行調(diào)節(jié)。免疫球蛋白樣轉(zhuǎn)錄子2（ILT2，又稱CD85j，LILRB1，LIR-1）是一種I型轉(zhuǎn)膜蛋白，胞漿區(qū)含有四個(gè)免疫受體酪氨酸抑制性基序。該受體通過動(dòng)員如SHP-1的Src同源結(jié)構(gòu)域（SH2）蛋白參與ITIM酪氨酸磷酸化的負(fù)信號調(diào)節(jié)。ILT2在NK細(xì)胞、T細(xì)胞、B細(xì)胞、單核細(xì)胞和樹突細(xì)胞上均有表達(dá)，與MHC-I類分子（HLA-A, HLA-B, HLA-C,HLA-E和HLA-G）結(jié)合，同時(shí)，ILT-2還能識別其他蛋白，，如人類巨細(xì)胞病毒（HCMV）編碼的UL18蛋白。目前，ILT2在抗結(jié)核感染免疫中的作用尚不明確。為此，本論文開展了對ILT2在結(jié)核患者NK細(xì)胞的表達(dá)及作用的研究： 第一，研究NK細(xì)胞及亞群在肺結(jié)核患者外周血淋巴細(xì)胞的分布情況，研究ILT2在肺結(jié)核患者NK細(xì)胞上的表達(dá)及與結(jié)核病情嚴(yán)重程度的關(guān)系。研究對象包含肺結(jié)核組（n=70）和健康對照組（n=67），根據(jù)痰涂片或痰培養(yǎng)結(jié)果將肺結(jié)核組分為菌陽組（n=41）和菌陰組肺結(jié)核組（n=29）。 首先采集活動(dòng)性肺結(jié)核組和健康對照組的新鮮外周EDTA抗凝血,密度梯度離心分離PBMCs，用熒光標(biāo)記的抗人CD3、CD56、CD16進(jìn)行染色，流式細(xì)胞術(shù)檢測CD3-CD56+NK細(xì)胞及兩個(gè)亞型CD3-CD56dimCD16+和CD3-CD56brightCD16+/-的分布比例。研究結(jié)果顯示，活動(dòng)性肺結(jié)核患者外周血淋巴細(xì)胞群中CD3-CD56+NK細(xì)胞的比例為2.3%(1.2%-4.0%)，明顯低于健康對照組（7.8%；6.5%-11.9%)，p0.0001；活動(dòng)性肺結(jié)核外周血淋巴細(xì)胞群中CD3-CD56dimNK細(xì)胞的比例(3.6%；2.2-6.7%)明顯低于健康對照組（9.2%；6.2-14.1%；p0.0001），活動(dòng)性肺結(jié)核組外周血淋巴細(xì)胞群中CD3-CD56brightNK細(xì)胞的比例(0.3%；0.2-0.6%)較健康對照組（0.5%；0.3-0.6%）顯著下降（p=0.0002）。研究結(jié)果表明活動(dòng)性肺結(jié)核患者外周血NK細(xì)胞及亞群的比例均明顯下降。 其次，采集肺結(jié)核組和健康對照組的新鮮外周EDTA抗凝血,密度梯度離心分離PBMCs，用熒光標(biāo)記的抗人CD3、CD56、CD16和ILT2染色，流式細(xì)胞術(shù)檢測ILT2抗體在NK細(xì)胞不同亞群上的表達(dá)。實(shí)驗(yàn)結(jié)果顯示，活動(dòng)性肺結(jié)核患者ILT2+CD56dimCD16+NK細(xì)胞比例45.7%(33.9%-56.2%)明顯高于健康對照者19.1%(9.9-30.0%)，(p0.0001)。ILT2在活動(dòng)性肺結(jié)核組CD56brightCD16+/-NK細(xì)胞上的表達(dá)比例為2.6%（1.4-4.9%)，與健康對照組CD56brightCD16+/-NK細(xì)胞表達(dá)的比例2.4%（0.7-5.7%)相似，結(jié)果無統(tǒng)計(jì)學(xué)意義(p=0.5163)。研究結(jié)果表明活動(dòng)性肺結(jié)核患者外周血ILT2+CD56dimCD16+NK細(xì)胞表達(dá)比例明顯高于健康對照者。 此外，比較了ILT2在菌陽活動(dòng)性肺結(jié)核組和菌陰活動(dòng)性肺結(jié)核組D56dimCD16+-NK細(xì)胞上的表達(dá)，分析ILT2在肺結(jié)核患者NK細(xì)胞上的表達(dá)與結(jié)核病病情的關(guān)系。實(shí)驗(yàn)結(jié)果顯示，ILT2在菌陽肺結(jié)核組CD56dimCD16+NK細(xì)胞上的表達(dá)比例(55.3%；45.4-59.9%)明顯高于菌陰肺結(jié)核組(32.9%；23.3-42.0%)， p0.0001。實(shí)驗(yàn)結(jié)果表明ILT2在CD56dimCD16+NK細(xì)胞上的表達(dá)水平與肺結(jié)核病的病情嚴(yán)重程度相關(guān)。 第二，通過研究ILT2表達(dá)與NK細(xì)胞CD107a表達(dá)的關(guān)系及ILT2表達(dá)與NK細(xì)胞分泌IFN-γ的關(guān)系，探究ILT2對肺結(jié)核患者NK細(xì)胞的細(xì)胞毒作用和干擾素分泌的影響。同時(shí)通過檢測ILT2+CD56dimNK細(xì)胞的自然凋亡與ILT2-CD56dimNK細(xì)胞的自然凋亡以研究ILT2對NK細(xì)胞的自然凋亡作用。研究對象為38例活動(dòng)性結(jié)核患者，痰涂片或痰培養(yǎng)結(jié)果陽性。 首先，以K562細(xì)胞為靶細(xì)胞，以5:1比例將15例活動(dòng)性結(jié)核患者外周血單個(gè)核細(xì)胞與靶細(xì)胞K562共孵育，加入抗人CD107a熒光抗體。采用流式細(xì)胞術(shù)分析CD107a在ILT2+CD56dimNK和ILT2-CD56dimNK細(xì)胞上的表達(dá)區(qū)別。實(shí)驗(yàn)結(jié)果顯示，CD107a在ILT2+CD56dimCD16+NK細(xì)胞的表達(dá)比例顯著低于ILT2-CD56dimNK細(xì)胞(p=0.0169)。結(jié)果表明ILT2在CD56dimNK細(xì)胞的表達(dá)與NK細(xì)胞細(xì)胞毒作用缺陷相關(guān)。 其次，采用胞內(nèi)染色方法，將10例活動(dòng)性肺結(jié)核患者單個(gè)核細(xì)胞與靶細(xì)胞K562混合孵育，加入monensin，細(xì)胞表面熒光染色后，行細(xì)胞破膜，加入FITC-標(biāo)記抗人IFN-γ單抗，流式細(xì)胞術(shù)檢測IFN-γ在ILT2+CD56dimNK細(xì)胞和ILT2-CD56dimNK細(xì)胞內(nèi)的表達(dá)。實(shí)驗(yàn)結(jié)果顯示，IFN-在ILT2+CD56dimNK細(xì)胞的表達(dá)明顯少于ILT2-CD56dimNK細(xì)胞（p=0.0038）。結(jié)果表明ILT2在CD56dimNK細(xì)胞的表達(dá)與NK細(xì)胞IFN-γ分泌水平降低相關(guān)。 此外，加入抗ILT2的阻斷抗體及同型對照抗體，將CD56dimCD16+NK細(xì)胞與靶細(xì)胞K562進(jìn)行孵育，流式細(xì)胞術(shù)檢測阻斷ILT2信號通路后CD107a在CD56dimCD16+NK細(xì)胞上表達(dá)情況。實(shí)驗(yàn)結(jié)果顯示，阻斷ILT2信號通路后，CD107a在CD56dimCD16+NK細(xì)胞上表達(dá)的比例較加入同型對照后明顯升高（p=0.0223）。結(jié)果表明阻斷ILT2信號通路可提高CD56dimCD16+NK細(xì)胞的細(xì)胞毒作用。 最后，采用Annexin V與PI雙染方法判定NK細(xì)胞自然凋亡，采用流式細(xì)胞術(shù)檢測，比較分析ILT2+CD56dimNK細(xì)胞和ILT2-CD56dimNK細(xì)胞的凋亡比率。實(shí)驗(yàn)結(jié)果顯示，ILT2+CD56dimNK細(xì)胞的自然凋亡率顯著高于ILT2-CD56dimNK細(xì)胞(p=0.0335)。研究結(jié)果表明ILT2可促進(jìn)活動(dòng)性肺結(jié)核患者CD56dimCD16+NK細(xì)胞的自然凋亡。 本研究采用流式細(xì)胞術(shù),開展了對ILT2在結(jié)核患者NK細(xì)胞的表達(dá)及作用的研究。研究發(fā)現(xiàn)，活動(dòng)性肺結(jié)核患者外周血中NK細(xì)胞及亞群的分布比例均顯著下降；ILT2在活動(dòng)性肺結(jié)核患者CD56dimCD16+NK細(xì)胞上的表達(dá)頻率升高，并與活動(dòng)性結(jié)核病的病情嚴(yán)重程度相關(guān)；ILT2對活動(dòng)性結(jié)核患者CD56dimNK細(xì)胞的細(xì)胞毒作用和細(xì)胞因子分泌的功能具有抑制作用；ILT2可促進(jìn)活動(dòng)性肺結(jié)核患者CD3-CD56dimNK細(xì)胞的自然凋亡。本研究為更深入了解NK細(xì)胞抗結(jié)核感染免疫理論，建立基于增強(qiáng)NK細(xì)胞功能的抗結(jié)核免疫治療提供了新的思路。
[Abstract]:Tuberculosis is the second leading cause of death. Due to the emergence of drug-resistant tuberculosis, the epidemic of AIDS infection, population mobility and the increasing use of clinical immunosuppressants, the global epidemic of tuberculosis is very serious. In 2012 alone, 8.6 million new cases of tuberculosis worldwide, 1.3 million people died of tuberculosis, about 450,000 people are resistant to tuberculosis. There are about 5 million active tuberculosis patients in China, and about 130,000 people die of tuberculosis every year. Despite the large number of tuberculosis infections, only 5-10% of the infected people develop into active tuberculosis. The results show that the occurrence, development and outcomes of tuberculosis not only depend on the number and virulence of Mycobacterium tuberculosis. To a large extent, it also depends on the immune state of the body.
The innate immune system plays an important role in the control and clearance of Mycobacterium tuberculosis before the acquired immunity is fully activated in the early stage of the invasion of Mycobacterium tuberculosis. Inherent immune cells are mainly NK cells, macrophages, monocytes, neutrophils, etc. They do not need antigen pre-sensitization, mainly through the release of cytokines, perforin, granzymes, expression ligands and other functions, induce immune cells to aggregate, kill target cells.
NK cells are important cells involved in the innate immunity against tuberculosis. They have cytotoxic effects, secrete cytokines such as IFN-gamma, and have immunoregulatory functions. In order to maintain the balance of anti-infective immunity, NK cells are regulated by different activation and inhibition receptors located on the cell surface. (ILT2, also known as CD85j, LILRB1, LIR-1) is a type I transmembrane protein with four tyrosine inhibitory motifs in the cytoplasm. The receptor is involved in negative signal regulation of ITIM tyrosine phosphorylation by mobilizing Src homologous domain (SH2) proteins such as SHP-1. ILT2 is expressed in NK cells, T cells, B cells, monocytes and dendritic cells. It binds to MHC-I molecules (HLA-A, HLA-B, HLA-C, HLA-E and HLA-G) and recognizes other proteins, such as UL18 protein encoded by human cytomegalovirus (HCMV). At present, the role of ILT2 in anti-tuberculosis immunity is not clear.
Firstly, the distribution of NK cells and their subsets in peripheral blood lymphocytes of pulmonary tuberculosis patients was studied, and the expression of ILT2 in NK cells of pulmonary tuberculosis patients and its relationship with the severity of tuberculosis were studied. =41) and pulmonary tuberculosis group (n=29).
Fresh peripheral EDTA anticoagulants were collected from active pulmonary tuberculosis group and healthy control group. PBMCs were separated by density gradient centrifugation. Anti-human CD3, CD56 and CD16 were stained with fluorescent labels. The distribution of CD3-CD56 + NK cells and two subtypes of CD3-CD56 dimCD16 + and CD3-CD56 bright CD16 + /- were detected by flow cytometry. The percentage of CD3-CD56+NK cells in peripheral blood lymphocyte population of patients with pulmonary tuberculosis was 2.3% (1.2% - 4.0%) significantly lower than that of healthy control group (7.8%; 6.5% - 11.9%) and p0.0001; the percentage of CD3-CD56 dim NK cells in peripheral blood lymphocyte population of active pulmonary tuberculosis (3.6%; 2.2-6.7%) was significantly lower than that of healthy control group (9.2%; 6.2-14.1%; p0.0001), and the activity of active pulmonary tuberculosis The percentage of CD3-CD56 bright NK cells (0.3%; 0.2-0.6%) in peripheral blood lymphocyte population of tuberculosis group was significantly lower than that of healthy control group (0.5%; 0.3-0.6%) (p = 0.0002). The results showed that the percentage of NK cells and subsets in peripheral blood of active pulmonary tuberculosis patients was significantly decreased.
Secondly, fresh peripheral EDTA anticoagulants were collected from pulmonary tuberculosis group and healthy control group, and PBMCs were separated by density gradient centrifugation. The expression of ILT2 antibody in different subsets of NK cells was detected by fluorescent staining of anti-human CD3, CD56, CD16 and ILT2. The results showed that the proportion of ILT2 + CD56dimCD16 + NK cells in active pulmonary tuberculosis patients was 45.7%. (33.9% - 56.2%) was significantly higher than that of healthy controls (19.1% (9.9-30.0%) (p0.0001). The expression of ILT2 on CD56 bright CD16 + / - NK cells was 2.6% (1.4-4.9%) in active pulmonary tuberculosis group and 2.4% (0.7-5.7%) in healthy control group. The results showed no statistical significance (p = 0.5163). The expression ratio of ILT2+CD56dimCD16+NK cells in peripheral blood of patients with pulmonary tuberculosis was significantly higher than that of healthy controls.
In addition, the expression of ILT2 on D56dimCD16 + - NK cells in bacterial positive active pulmonary tuberculosis group and bacterial negative active pulmonary tuberculosis group was compared, and the relationship between the expression of ILT2 on NK cells and tuberculosis was analyzed. The expression level of ILT2 on CD56 dim CD16 + NK cells was correlated with the severity of tuberculosis.
Secondly, by studying the relationship between the expression of ILT2 and the expression of CD107a in NK cells, and the relationship between the expression of ILT2 and the secretion of IFN-gamma in NK cells, the cytotoxic effect of ILT2 on NK cells and the secretion of interferon were investigated. The natural apoptosis of cells was observed in 38 patients with active tuberculosis. The results of sputum smear or sputum culture were positive.
Firstly, the peripheral blood mononuclear cells of 15 active tuberculosis patients were co-incubated with the target cell K562 at a ratio of 5:1 and added with anti-human CD107a fluorescent antibody. The expression of CD107a in ILT2+CD56dimNK and ILT2-CD56dim NK cells was analyzed by flow cytometry. The expression of ILT2 in CD56dim NK cells was significantly lower than that in ILT2-CD56dim NK cells (p=0.0169).
Secondly, the mononuclear cells of 10 patients with active pulmonary tuberculosis were incubated with target cell K562 by intracellular staining. Monensin was added to the cells. After fluorescence staining, the membrane was broken and FITC-labeled anti-human IFN-gamma monoclonal antibody was added. The expression of IFN-gamma in ILT2+CD56dim NK cells and ILT2-CD56dim NK cells was detected by flow cytometry. The results showed that the expression of IFN-in ILT2+CD56dim NK cells was significantly lower than that in ILT2-CD56dim NK cells (p=0.0038). The results showed that the expression of ILT2 in CD56dim NK cells was correlated with the decrease of IFN-gamma secretion of NK cells.
In addition, CD56 dimCD16 + NK cells were incubated with K562 cells by adding anti-ILT2 blocking antibodies and homo-control antibodies. The expression of CD107a on CD56 dimCD16 + NK cells was detected by flow cytometry after blocking the ILT2 signaling pathway. The results showed that the expression of CD107a on CD56 dimCD16 + NK cells was higher after blocking the ILT2 signaling pathway. The results showed that blocking ILT2 signaling pathway could enhance the cytotoxicity of CD56 dim CD16 + NK cells.
Finally, the natural apoptosis of NK cells was determined by Annexin V staining and PI staining. The apoptosis rates of ILT2+CD56dim NK cells and ILT2-CD56dim NK cells were compared and analyzed by flow cytometry. The results showed that the natural apoptosis rate of ILT2+CD56dim NK cells was significantly higher than that of ILT2-CD56dim NK cells (p=0.0335). Spontaneous apoptosis of CD56dimCD16+NK cells in patients with active pulmonary tuberculosis.
In this study, flow cytometry was used to study the expression and role of ILT2 in NK cells of tuberculosis patients. The results showed that the distribution of NK cells and subsets in peripheral blood of active pulmonary tuberculosis patients were significantly decreased; the expression of ILT2 in CD56 dimCD16 + NK cells of active pulmonary tuberculosis patients was increased, and it was associated with active tuberculosis. The severity of the disease is related; ILT2 inhibits the cytotoxicity and cytokine secretion of CD56dim NK cells in active tuberculosis patients; ILT2 promotes the natural apoptosis of CD3-CD56dim NK cells in active tuberculosis patients. Cell function provides a new idea for anti TB immunotherapy.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R52
本文編號：2178044
[Abstract]:Tuberculosis is the second leading cause of death. Due to the emergence of drug-resistant tuberculosis, the epidemic of AIDS infection, population mobility and the increasing use of clinical immunosuppressants, the global epidemic of tuberculosis is very serious. In 2012 alone, 8.6 million new cases of tuberculosis worldwide, 1.3 million people died of tuberculosis, about 450,000 people are resistant to tuberculosis. There are about 5 million active tuberculosis patients in China, and about 130,000 people die of tuberculosis every year. Despite the large number of tuberculosis infections, only 5-10% of the infected people develop into active tuberculosis. The results show that the occurrence, development and outcomes of tuberculosis not only depend on the number and virulence of Mycobacterium tuberculosis. To a large extent, it also depends on the immune state of the body.
The innate immune system plays an important role in the control and clearance of Mycobacterium tuberculosis before the acquired immunity is fully activated in the early stage of the invasion of Mycobacterium tuberculosis. Inherent immune cells are mainly NK cells, macrophages, monocytes, neutrophils, etc. They do not need antigen pre-sensitization, mainly through the release of cytokines, perforin, granzymes, expression ligands and other functions, induce immune cells to aggregate, kill target cells.
NK cells are important cells involved in the innate immunity against tuberculosis. They have cytotoxic effects, secrete cytokines such as IFN-gamma, and have immunoregulatory functions. In order to maintain the balance of anti-infective immunity, NK cells are regulated by different activation and inhibition receptors located on the cell surface. (ILT2, also known as CD85j, LILRB1, LIR-1) is a type I transmembrane protein with four tyrosine inhibitory motifs in the cytoplasm. The receptor is involved in negative signal regulation of ITIM tyrosine phosphorylation by mobilizing Src homologous domain (SH2) proteins such as SHP-1. ILT2 is expressed in NK cells, T cells, B cells, monocytes and dendritic cells. It binds to MHC-I molecules (HLA-A, HLA-B, HLA-C, HLA-E and HLA-G) and recognizes other proteins, such as UL18 protein encoded by human cytomegalovirus (HCMV). At present, the role of ILT2 in anti-tuberculosis immunity is not clear.
Firstly, the distribution of NK cells and their subsets in peripheral blood lymphocytes of pulmonary tuberculosis patients was studied, and the expression of ILT2 in NK cells of pulmonary tuberculosis patients and its relationship with the severity of tuberculosis were studied. =41) and pulmonary tuberculosis group (n=29).
Fresh peripheral EDTA anticoagulants were collected from active pulmonary tuberculosis group and healthy control group. PBMCs were separated by density gradient centrifugation. Anti-human CD3, CD56 and CD16 were stained with fluorescent labels. The distribution of CD3-CD56 + NK cells and two subtypes of CD3-CD56 dimCD16 + and CD3-CD56 bright CD16 + /- were detected by flow cytometry. The percentage of CD3-CD56+NK cells in peripheral blood lymphocyte population of patients with pulmonary tuberculosis was 2.3% (1.2% - 4.0%) significantly lower than that of healthy control group (7.8%; 6.5% - 11.9%) and p0.0001; the percentage of CD3-CD56 dim NK cells in peripheral blood lymphocyte population of active pulmonary tuberculosis (3.6%; 2.2-6.7%) was significantly lower than that of healthy control group (9.2%; 6.2-14.1%; p0.0001), and the activity of active pulmonary tuberculosis The percentage of CD3-CD56 bright NK cells (0.3%; 0.2-0.6%) in peripheral blood lymphocyte population of tuberculosis group was significantly lower than that of healthy control group (0.5%; 0.3-0.6%) (p = 0.0002). The results showed that the percentage of NK cells and subsets in peripheral blood of active pulmonary tuberculosis patients was significantly decreased.
Secondly, fresh peripheral EDTA anticoagulants were collected from pulmonary tuberculosis group and healthy control group, and PBMCs were separated by density gradient centrifugation. The expression of ILT2 antibody in different subsets of NK cells was detected by fluorescent staining of anti-human CD3, CD56, CD16 and ILT2. The results showed that the proportion of ILT2 + CD56dimCD16 + NK cells in active pulmonary tuberculosis patients was 45.7%. (33.9% - 56.2%) was significantly higher than that of healthy controls (19.1% (9.9-30.0%) (p0.0001). The expression of ILT2 on CD56 bright CD16 + / - NK cells was 2.6% (1.4-4.9%) in active pulmonary tuberculosis group and 2.4% (0.7-5.7%) in healthy control group. The results showed no statistical significance (p = 0.5163). The expression ratio of ILT2+CD56dimCD16+NK cells in peripheral blood of patients with pulmonary tuberculosis was significantly higher than that of healthy controls.
In addition, the expression of ILT2 on D56dimCD16 + - NK cells in bacterial positive active pulmonary tuberculosis group and bacterial negative active pulmonary tuberculosis group was compared, and the relationship between the expression of ILT2 on NK cells and tuberculosis was analyzed. The expression level of ILT2 on CD56 dim CD16 + NK cells was correlated with the severity of tuberculosis.
Secondly, by studying the relationship between the expression of ILT2 and the expression of CD107a in NK cells, and the relationship between the expression of ILT2 and the secretion of IFN-gamma in NK cells, the cytotoxic effect of ILT2 on NK cells and the secretion of interferon were investigated. The natural apoptosis of cells was observed in 38 patients with active tuberculosis. The results of sputum smear or sputum culture were positive.
Firstly, the peripheral blood mononuclear cells of 15 active tuberculosis patients were co-incubated with the target cell K562 at a ratio of 5:1 and added with anti-human CD107a fluorescent antibody. The expression of CD107a in ILT2+CD56dimNK and ILT2-CD56dim NK cells was analyzed by flow cytometry. The expression of ILT2 in CD56dim NK cells was significantly lower than that in ILT2-CD56dim NK cells (p=0.0169).
Secondly, the mononuclear cells of 10 patients with active pulmonary tuberculosis were incubated with target cell K562 by intracellular staining. Monensin was added to the cells. After fluorescence staining, the membrane was broken and FITC-labeled anti-human IFN-gamma monoclonal antibody was added. The expression of IFN-gamma in ILT2+CD56dim NK cells and ILT2-CD56dim NK cells was detected by flow cytometry. The results showed that the expression of IFN-in ILT2+CD56dim NK cells was significantly lower than that in ILT2-CD56dim NK cells (p=0.0038). The results showed that the expression of ILT2 in CD56dim NK cells was correlated with the decrease of IFN-gamma secretion of NK cells.
In addition, CD56 dimCD16 + NK cells were incubated with K562 cells by adding anti-ILT2 blocking antibodies and homo-control antibodies. The expression of CD107a on CD56 dimCD16 + NK cells was detected by flow cytometry after blocking the ILT2 signaling pathway. The results showed that the expression of CD107a on CD56 dimCD16 + NK cells was higher after blocking the ILT2 signaling pathway. The results showed that blocking ILT2 signaling pathway could enhance the cytotoxicity of CD56 dim CD16 + NK cells.
Finally, the natural apoptosis of NK cells was determined by Annexin V staining and PI staining. The apoptosis rates of ILT2+CD56dim NK cells and ILT2-CD56dim NK cells were compared and analyzed by flow cytometry. The results showed that the natural apoptosis rate of ILT2+CD56dim NK cells was significantly higher than that of ILT2-CD56dim NK cells (p=0.0335). Spontaneous apoptosis of CD56dimCD16+NK cells in patients with active pulmonary tuberculosis.
In this study, flow cytometry was used to study the expression and role of ILT2 in NK cells of tuberculosis patients. The results showed that the distribution of NK cells and subsets in peripheral blood of active pulmonary tuberculosis patients were significantly decreased; the expression of ILT2 in CD56 dimCD16 + NK cells of active pulmonary tuberculosis patients was increased, and it was associated with active tuberculosis. The severity of the disease is related; ILT2 inhibits the cytotoxicity and cytokine secretion of CD56dim NK cells in active tuberculosis patients; ILT2 promotes the natural apoptosis of CD3-CD56dim NK cells in active tuberculosis patients. Cell function provides a new idea for anti TB immunotherapy.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R52
本文編號：2178044
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