骨髓增生異常綜合征患者外周血NK細(xì)胞數(shù)量和功能的研究
發(fā)布時(shí)間：2018-07-18 15:01
【摘要】：目的研究MDS患者與正常人外周血NK細(xì)胞的數(shù)量、亞型、功能分子和殺傷功能的變化;探討NK細(xì)胞在MDS的發(fā)病和疾病進(jìn)展中所起的作用,也許可以為將來(lái)MDS的細(xì)胞和靶向治療提供理論依據(jù)。背景骨髓增生異常綜合征(Myelodysplastic syndromes,MDS)是一組起源于骨髓造血干細(xì)胞的惡性克隆性血液系統(tǒng)腫瘤,以骨髓髓系細(xì)胞發(fā)育異常、克隆性無(wú)效造血、病態(tài)造血進(jìn)而導(dǎo)致的外周血一系或多系血細(xì)胞減少為特點(diǎn),大約1/3的MDS患者最終進(jìn)展為急性髓系白血病(Acute myelogenous leukemia,AML)。自然殺傷細(xì)胞(Natural killer cells,NK細(xì)胞)是淋巴細(xì)胞的一種,在正常人外周血淋巴細(xì)胞中所占比例約為10%-15%,具有早期可產(chǎn)生細(xì)胞因子、趨化因子和非致敏即可溶解靶細(xì)胞的能力,因此在機(jī)體抗腫瘤、抗病毒免疫中具有重要作用。近年來(lái),人們逐漸發(fā)現(xiàn)MDS患者中NK細(xì)胞數(shù)量和功能均存在異常,并與MDS的病情和疾病進(jìn)展具有相關(guān)性。本研究應(yīng)用流式細(xì)胞術(shù)檢測(cè)MDS患者外周血NK細(xì)胞的數(shù)量、亞型和受體的表達(dá),應(yīng)用共培養(yǎng)的方法檢測(cè)NK細(xì)胞對(duì)靶細(xì)胞的殺傷功能,并與MDS患者的的病情和相關(guān)臨床指標(biāo)進(jìn)行相關(guān)性分析。方法收集天津醫(yī)科大學(xué)總醫(yī)院自2015年10月至2016年6月收治的35例MDS患者及34名正常對(duì)照者的外周血標(biāo)本。第一部分應(yīng)用流式細(xì)胞術(shù)檢測(cè)MDS患者和正常對(duì)照者外周血NK細(xì)胞(CD3-CD56+)、CD56brightNK細(xì)胞(CD3-CD56brightCD16-)、CD56dimNK細(xì)胞(CD3-CD56dimCD16+)、T細(xì)胞(CD3+)、NKT細(xì)胞(CD3+CD56+)的數(shù)量,NK細(xì)胞的兩個(gè)亞型即CD56brightNK細(xì)胞、CD56dimNK細(xì)胞的構(gòu)成比及CD56brightNK細(xì)胞/CD56dimNK細(xì)胞;檢測(cè)NK細(xì)胞表面的功能分子NKp30、NKp46、NKG2A的表達(dá)情況;檢測(cè)MDS患者治療前后NK細(xì)胞的數(shù)量和功能分子有無(wú)變化;根據(jù)MDS不同危險(xiǎn)度分層進(jìn)行分析;并與骨髓分類中骨髓原始細(xì)胞百分比、外周血中性粒細(xì)胞絕對(duì)值(ANC)、血紅蛋白含量(Hb)做相關(guān)性分析。第二部分收集7例MDS患者和8名正常對(duì)照者新鮮抗凝外周血,應(yīng)用免疫磁珠法分選出CD3-CD56+NK細(xì)胞,體外應(yīng)用高濃度IL-2刺激過(guò)夜培養(yǎng)后與靶細(xì)胞(K562細(xì)胞)進(jìn)行共培養(yǎng),6h后,收獲細(xì)胞并標(biāo)記PI,應(yīng)用流式細(xì)胞術(shù)檢測(cè)NK細(xì)胞殺傷功能。結(jié)果第一部分(1)NK細(xì)胞數(shù)量:MDS組NK/Lym%、CD56dimNK/Lym%顯著低于對(duì)照組,分別為(8.19±5.30 vs 13.81±5.96,P0.001)、(7.95±5.14 vs 12.78±5.74,P=0.001);MDS組CD56brightNK/Lym%高于對(duì)照組(0.68±0.33 vs 0.53±0.22,P0.05);MDS組NKT/Lym%、T cells/Lym%與對(duì)照組無(wú)顯著差異,分別為(1.46±1.30 vs 1.87±1.33,P=0.198)和(66.70±16.38 vs 67.09±9.90,P=0.907);分析NK細(xì)胞亞型,MDS組CD56dimNK/NK%明顯低于對(duì)照組(91.12±3.49 vs 95.40±2.48,P0.001);CD56brightNK/NK%明顯高于對(duì)照組(8.87±3.49 vs 4.60±2.48,P0.001);MDS組CD56brightNK/CD56dimNK%細(xì)胞明顯高于對(duì)照組(9.90±4.29 vs4.89±2.77,P0.001);MDS組NK細(xì)胞、CD56dimNK、CD56brightNK細(xì)胞的絕對(duì)值(/ul)均低于對(duì)照組,分別為(125.16±94.39 vs 295.86±119.12,P0.0001)、(127.88±103.00 vs 281.34±136.53,P0.0001),(10.83±4.53 vs 6.24±4.66,P0.001)。(2)NK細(xì)胞表面功能分子的表達(dá):MDS組NK細(xì)胞表面NKG2A的表達(dá)與對(duì)照組無(wú)明顯差異(37.31±19.60 vs 37.45±14.24,P㧐0.05);而活化性受體NKp30和NKp46的表達(dá)明顯低于對(duì)照組,分別為(74.35±15.36 vs 84.89±8.73,P=0.001)、(77.79±15.30 vs 89.63±9.12,P0.001)。(3)3例MDS患者治療后,隨著病情好轉(zhuǎn),NK/Lym%、CD56dimNK/Lym%逐漸恢復(fù),隨著病情進(jìn)展,NK細(xì)胞數(shù)量進(jìn)一步下降。(4)在MDS組中,高危組NK/Lym%、CD56dimNK/Lym%明顯低于低危組(5.41±0.97 vs 10.13±1.83,P=0.04)、(4.64±0.94 vs 8.81±1.14,P=0.02);而高危組與低危組NKG2A、NKp30、NKp46的表達(dá)無(wú)明顯差異,分別為(31.97±2.74 vs 44.71±9.16,P=0.16)、(71.26±5.11 vs 77.44±6.81,P=0.47)、(75.70±6.46 vs 78.70±5.28,P=0.74)。(5)MDS患者NK細(xì)胞數(shù)量與功能分子的表達(dá)與臨床指標(biāo)的相關(guān)性:MDS患者NK/Lym%、CD56dimNK/Lym%與患者骨髓原始細(xì)胞比例呈負(fù)相關(guān)(r=-0.53和-0.68,P0.05),與外周血血紅蛋白含量(Hb)呈正相關(guān)(r=0.35和0.50,P0.05);與中性粒細(xì)胞絕對(duì)值(ANC)呈正相關(guān)(r=0.52和0.53,P0.01);NK細(xì)胞表面活化性受體NKp46的表達(dá)與骨髓原始細(xì)胞數(shù)呈負(fù)相關(guān)(r=-0.584,P0.05)。第二部分NK細(xì)胞與K562共培養(yǎng)后,MDS組K562細(xì)胞的凋亡率低于對(duì)照組(7.73±4.0 vs 3.14±2.47,P=0.029)。結(jié)論:MDS患者NK細(xì)胞數(shù)量減少,亞型失衡,活化性受體下降,可能導(dǎo)致其活化不足,殺傷功能下降,從而導(dǎo)致其不能正常行使有效的免疫監(jiān)視功能,進(jìn)而不能早期有效清除MDS惡性克隆細(xì)胞,導(dǎo)致MDS患者病情進(jìn)展。
[Abstract]:Objective to study the changes in the number, subtypes, functional molecules and killing function of NK cells in peripheral blood of MDS patients and normal people, and to explore the role of NK cells in the pathogenesis and disease progression of MDS, and may provide a theoretical basis for the cell and target therapy of MDS in the future. Background myelodysplastic syndrome (Myelodysplastic syndromes, MDS). A group of malignant clonal hematological tumors originated from bone marrow hematopoietic stem cells, characterized by abnormal development of marrow myeloid cells, clonogenic ineffective hematopoiesis, pathological hematopoiesis and hematopoiesis resulting in peripheral blood cells or multilineage blood cells, and about 1/3 of MDS patients eventually progressed to acute myeloid leukemia (Acute myelogenous leukemia, AML). Natural killer cells (Natural killer cells, NK cells) are a kind of lymphocyte, and the proportion of peripheral blood lymphocytes in normal human peripheral blood lymphocytes is about 10%-15%. It has the ability to produce cytokines, chemokines and non sensitizing target cells in the early stage. Therefore, it plays an important role in the anti-tumor and antiviral immunity of the body. It is gradually found that the number and function of NK cells in MDS patients are abnormal, and are related to the condition of MDS and the progression of the disease. This study used flow cytometry to detect the number of NK cells in peripheral blood of MDS patients, the expression of subtypes and receptors, and the use of CO culture to detect the killing function of NK cells to the target cells, and with the MDS patients. Methods the peripheral blood samples of 35 MDS patients and 34 normal controls were collected from October 2015 to June 2016 in General Hospital Affiliated to Tianjin Medical University. The first part used flow cytometry to detect the peripheral blood NK cells (CD3-CD56+) and CD56brightNK cells (CD56brightNK cells) in MDS patients and normal controls. CD3-CD56brightCD16-), the number of CD56dimNK cells (CD3-CD56dimCD16+), T cells (CD3+), and NKT cells (CD3+CD56+), the two subtypes of NK cells, the CD56brightNK cells, the constituent ratio of CD56dimNK cells and the CD56brightNK cell /CD56dimNK cells. The number and functional molecules of NK cells were changed before and after. According to the different risk levels of MDS, the percentage of bone marrow cells in the bone marrow, the absolute value of peripheral blood neutrophils (ANC) and the content of hemoglobin (Hb) were analyzed. The second part collected the fresh anticoagulant peripheral blood of 7 MDS patients and 8 normal controls. CD3-CD56+NK cells were selected by immunomagnetic beads. In vitro, high concentration IL-2 stimulation was used to co culture with target cells (K562 cells). After 6h, the harvested cells were labeled with PI, and the cytotoxic function of NK cells was detected by flow cytometry. Results the number one (1) NK fine cell number: MDS group NK/Lym%, CD56dimNK/Lym% significantly lower than the control group. (8.19 + 5.30 vs 13.81 + 5.96, P0.001), (7.95 + 5.14 vs 12.78 + 5.74, P=0.001), CD56brightNK/Lym% in MDS group was higher than that of control group (0.68 + 0.33 vs 0.53 + 0.22, P0.05), MDS group NKT/Lym%, T cells/Lym% and control group, respectively. The cell subtype, MDS group CD56dimNK/NK% was significantly lower than that of the control group (91.12 + 3.49 vs 95.40 + 2.48, P0.001), CD56brightNK/NK% was significantly higher than that of the control group (8.87 + 3.49 vs 4.60 + 2.48, P0.001), and MDS group CD56brightNK/CD56dimNK% cells were significantly higher than the control group (9.90 + 4.29 vs4.89 + 2.77, P0.001). The value (/ul) was lower than that of the control group (125.16 + 94.39 vs 295.86 + 119.12, P0.0001), (127.88 + 103 vs 281.34 + 136.53, P0.0001), (10.83 + 4.53 vs 6.24 + 4.66, P0.001). (2) the expression of functional molecules on the surface of NK cells: the expression of NKG2A on the MDS group NK cell surface was not significantly different from that of the control group. The expression of activated receptor NKp30 and NKp46 was significantly lower than that of the control group (74.35 + 15.36 vs 84.89 + 8.73, P=0.001), (77.79 + 15.30 vs 89.63 + 9.12, P0.001). (3) 3 cases of MDS patients were treated, NK/Lym%, CD56dimNK/Lym% gradually recovered with the improvement of the condition, and the number of NK cells decreased further as the condition progressed. (4) high risk in MDS group. Group NK/Lym%, CD56dimNK/Lym% was significantly lower than that of low risk group (5.41 + 0.97 vs 10.13 + 1.83, P=0.04), (4.64 + 0.94 vs 8.81 + 1.14, P=0.02), while the expression of NKG2A, NKp30 and NKp46 in high risk group and low risk group was (31.97 + 2.74 vs 44.71 + 9.16, P=0.16), respectively. 5) the correlation between the number of NK cells and the expression of functional molecules in MDS patients with the clinical indicators: NK/Lym% in MDS patients, CD56dimNK/Lym% was negatively correlated with the proportion of bone marrow cells (r=-0.53 and -0.68, P0.05), and was positively correlated with the content of hemoglobin (Hb) in peripheral blood (r=0.35 and 0.50, P0.05), and was positively correlated with the absolute value of neutrophils (ANC). 0.53, P0.01); the expression of NK cell surface activated receptor NKp46 was negatively correlated with the number of primitive cells in bone marrow (r=-0.584, P0.05). The apoptosis rate of K562 cells in MDS group was lower than that of the control group (7.73 + 4 vs 3.14 + 2.47, P=0.029). It may lead to the insufficiency of activation and the decrease of the killing function, which can lead to the failure to exercise the effective immune surveillance function, and thus can not effectively remove the MDS malignant clones early and effectively, and lead to the progression of MDS patients.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R551.3
本文編號(hào)：2132271
[Abstract]:Objective to study the changes in the number, subtypes, functional molecules and killing function of NK cells in peripheral blood of MDS patients and normal people, and to explore the role of NK cells in the pathogenesis and disease progression of MDS, and may provide a theoretical basis for the cell and target therapy of MDS in the future. Background myelodysplastic syndrome (Myelodysplastic syndromes, MDS). A group of malignant clonal hematological tumors originated from bone marrow hematopoietic stem cells, characterized by abnormal development of marrow myeloid cells, clonogenic ineffective hematopoiesis, pathological hematopoiesis and hematopoiesis resulting in peripheral blood cells or multilineage blood cells, and about 1/3 of MDS patients eventually progressed to acute myeloid leukemia (Acute myelogenous leukemia, AML). Natural killer cells (Natural killer cells, NK cells) are a kind of lymphocyte, and the proportion of peripheral blood lymphocytes in normal human peripheral blood lymphocytes is about 10%-15%. It has the ability to produce cytokines, chemokines and non sensitizing target cells in the early stage. Therefore, it plays an important role in the anti-tumor and antiviral immunity of the body. It is gradually found that the number and function of NK cells in MDS patients are abnormal, and are related to the condition of MDS and the progression of the disease. This study used flow cytometry to detect the number of NK cells in peripheral blood of MDS patients, the expression of subtypes and receptors, and the use of CO culture to detect the killing function of NK cells to the target cells, and with the MDS patients. Methods the peripheral blood samples of 35 MDS patients and 34 normal controls were collected from October 2015 to June 2016 in General Hospital Affiliated to Tianjin Medical University. The first part used flow cytometry to detect the peripheral blood NK cells (CD3-CD56+) and CD56brightNK cells (CD56brightNK cells) in MDS patients and normal controls. CD3-CD56brightCD16-), the number of CD56dimNK cells (CD3-CD56dimCD16+), T cells (CD3+), and NKT cells (CD3+CD56+), the two subtypes of NK cells, the CD56brightNK cells, the constituent ratio of CD56dimNK cells and the CD56brightNK cell /CD56dimNK cells. The number and functional molecules of NK cells were changed before and after. According to the different risk levels of MDS, the percentage of bone marrow cells in the bone marrow, the absolute value of peripheral blood neutrophils (ANC) and the content of hemoglobin (Hb) were analyzed. The second part collected the fresh anticoagulant peripheral blood of 7 MDS patients and 8 normal controls. CD3-CD56+NK cells were selected by immunomagnetic beads. In vitro, high concentration IL-2 stimulation was used to co culture with target cells (K562 cells). After 6h, the harvested cells were labeled with PI, and the cytotoxic function of NK cells was detected by flow cytometry. Results the number one (1) NK fine cell number: MDS group NK/Lym%, CD56dimNK/Lym% significantly lower than the control group. (8.19 + 5.30 vs 13.81 + 5.96, P0.001), (7.95 + 5.14 vs 12.78 + 5.74, P=0.001), CD56brightNK/Lym% in MDS group was higher than that of control group (0.68 + 0.33 vs 0.53 + 0.22, P0.05), MDS group NKT/Lym%, T cells/Lym% and control group, respectively. The cell subtype, MDS group CD56dimNK/NK% was significantly lower than that of the control group (91.12 + 3.49 vs 95.40 + 2.48, P0.001), CD56brightNK/NK% was significantly higher than that of the control group (8.87 + 3.49 vs 4.60 + 2.48, P0.001), and MDS group CD56brightNK/CD56dimNK% cells were significantly higher than the control group (9.90 + 4.29 vs4.89 + 2.77, P0.001). The value (/ul) was lower than that of the control group (125.16 + 94.39 vs 295.86 + 119.12, P0.0001), (127.88 + 103 vs 281.34 + 136.53, P0.0001), (10.83 + 4.53 vs 6.24 + 4.66, P0.001). (2) the expression of functional molecules on the surface of NK cells: the expression of NKG2A on the MDS group NK cell surface was not significantly different from that of the control group. The expression of activated receptor NKp30 and NKp46 was significantly lower than that of the control group (74.35 + 15.36 vs 84.89 + 8.73, P=0.001), (77.79 + 15.30 vs 89.63 + 9.12, P0.001). (3) 3 cases of MDS patients were treated, NK/Lym%, CD56dimNK/Lym% gradually recovered with the improvement of the condition, and the number of NK cells decreased further as the condition progressed. (4) high risk in MDS group. Group NK/Lym%, CD56dimNK/Lym% was significantly lower than that of low risk group (5.41 + 0.97 vs 10.13 + 1.83, P=0.04), (4.64 + 0.94 vs 8.81 + 1.14, P=0.02), while the expression of NKG2A, NKp30 and NKp46 in high risk group and low risk group was (31.97 + 2.74 vs 44.71 + 9.16, P=0.16), respectively. 5) the correlation between the number of NK cells and the expression of functional molecules in MDS patients with the clinical indicators: NK/Lym% in MDS patients, CD56dimNK/Lym% was negatively correlated with the proportion of bone marrow cells (r=-0.53 and -0.68, P0.05), and was positively correlated with the content of hemoglobin (Hb) in peripheral blood (r=0.35 and 0.50, P0.05), and was positively correlated with the absolute value of neutrophils (ANC). 0.53, P0.01); the expression of NK cell surface activated receptor NKp46 was negatively correlated with the number of primitive cells in bone marrow (r=-0.584, P0.05). The apoptosis rate of K562 cells in MDS group was lower than that of the control group (7.73 + 4 vs 3.14 + 2.47, P=0.029). It may lead to the insufficiency of activation and the decrease of the killing function, which can lead to the failure to exercise the effective immune surveillance function, and thus can not effectively remove the MDS malignant clones early and effectively, and lead to the progression of MDS patients.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R551.3
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