骨髓增生異常綜合征患者外周血NK細胞數(shù)量和功能的研究
發(fā)布時間：2018-07-18 15:01
【摘要】：目的研究MDS患者與正常人外周血NK細胞的數(shù)量、亞型、功能分子和殺傷功能的變化;探討NK細胞在MDS的發(fā)病和疾病進展中所起的作用,也許可以為將來MDS的細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胞的惡性克隆性血液系統(tǒng)腫瘤,以骨髓髓系細胞發(fā)育異常、克隆性無效造血、病態(tài)造血進而導致的外周血一系或多系血細胞減少為特點,大約1/3的MDS患者最終進展為急性髓系白血病(Acute myelogenous leukemia,AML)。自然殺傷細胞(Natural killer cells,NK細胞)是淋巴細胞的一種,在正常人外周血淋巴細胞中所占比例約為10%-15%,具有早期可產(chǎn)生細胞因子、趨化因子和非致敏即可溶解靶細胞的能力,因此在機體抗腫瘤、抗病毒免疫中具有重要作用。近年來,人們逐漸發(fā)現(xiàn)MDS患者中NK細胞數(shù)量和功能均存在異常,并與MDS的病情和疾病進展具有相關性。本研究應用流式細胞術檢測MDS患者外周血NK細胞的數(shù)量、亞型和受體的表達,應用共培養(yǎng)的方法檢測NK細胞對靶細胞的殺傷功能,并與MDS患者的的病情和相關臨床指標進行相關性分析。方法收集天津醫(yī)科大學總醫(yī)院自2015年10月至2016年6月收治的35例MDS患者及34名正常對照者的外周血標本。第一部分應用流式細胞術檢測MDS患者和正常對照者外周血NK細胞(CD3-CD56+)、CD56brightNK細胞(CD3-CD56brightCD16-)、CD56dimNK細胞(CD3-CD56dimCD16+)、T細胞(CD3+)、NKT細胞(CD3+CD56+)的數(shù)量,NK細胞的兩個亞型即CD56brightNK細胞、CD56dimNK細胞的構成比及CD56brightNK細胞/CD56dimNK細胞;檢測NK細胞表面的功能分子NKp30、NKp46、NKG2A的表達情況;檢測MDS患者治療前后NK細胞的數(shù)量和功能分子有無變化;根據(jù)MDS不同危險度分層進行分析;并與骨髓分類中骨髓原始細胞百分比、外周血中性粒細胞絕對值(ANC)、血紅蛋白含量(Hb)做相關性分析。第二部分收集7例MDS患者和8名正常對照者新鮮抗凝外周血,應用免疫磁珠法分選出CD3-CD56+NK細胞,體外應用高濃度IL-2刺激過夜培養(yǎng)后與靶細胞(K562細胞)進行共培養(yǎng),6h后,收獲細胞并標記PI,應用流式細胞術檢測NK細胞殺傷功能。結果第一部分(1)NK細胞數(shù)量:MDS組NK/Lym%、CD56dimNK/Lym%顯著低于對照組,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%高于對照組(0.68±0.33 vs 0.53±0.22,P0.05);MDS組NKT/Lym%、T cells/Lym%與對照組無顯著差異,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細胞亞型,MDS組CD56dimNK/NK%明顯低于對照組(91.12±3.49 vs 95.40±2.48,P0.001);CD56brightNK/NK%明顯高于對照組(8.87±3.49 vs 4.60±2.48,P0.001);MDS組CD56brightNK/CD56dimNK%細胞明顯高于對照組(9.90±4.29 vs4.89±2.77,P0.001);MDS組NK細胞、CD56dimNK、CD56brightNK細胞的絕對值(/ul)均低于對照組,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細胞表面功能分子的表達:MDS組NK細胞表面NKG2A的表達與對照組無明顯差異(37.31±19.60 vs 37.45±14.24,P㧐0.05);而活化性受體NKp30和NKp46的表達明顯低于對照組,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,NK/Lym%、CD56dimNK/Lym%逐漸恢復,隨著病情進展,NK細胞數(shù)量進一步下降。(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的表達無明顯差異,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細胞數(shù)量與功能分子的表達與臨床指標的相關性:MDS患者NK/Lym%、CD56dimNK/Lym%與患者骨髓原始細胞比例呈負相關(r=-0.53和-0.68,P0.05),與外周血血紅蛋白含量(Hb)呈正相關(r=0.35和0.50,P0.05);與中性粒細胞絕對值(ANC)呈正相關(r=0.52和0.53,P0.01);NK細胞表面活化性受體NKp46的表達與骨髓原始細胞數(shù)呈負相關(r=-0.584,P0.05)。第二部分NK細胞與K562共培養(yǎng)后,MDS組K562細胞的凋亡率低于對照組(7.73±4.0 vs 3.14±2.47,P=0.029)。結論:MDS患者NK細胞數(shù)量減少,亞型失衡,活化性受體下降,可能導致其活化不足,殺傷功能下降,從而導致其不能正常行使有效的免疫監(jiān)視功能,進而不能早期有效清除MDS惡性克隆細胞,導致MDS患者病情進展。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R551.3
本文編號：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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R551.3
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