骨髓增生异常综合征患者外周血NK细胞数量和功能的研究
发布时间：2018-07-18 15:01
【摘要】：目的研究MDS患者与正常人外周血NK细胞的数量、亚型、功能分子和杀伤功能的变化;探讨NK细胞在MDS的发病和疾病进展中所起的作用,也许可以为将来MDS的细胞和靶向治疗提供理论依据。背景骨髓增生异常综合征(Myelodysplastic syndromes,MDS)是一组起源于骨髓造血干细胞的恶性克隆性血液系统肿瘤,以骨髓髓系细胞发育异常、克隆性无效造血、病态造血进而导致的外周血一系或多系血细胞减少为特点,大约1/3的MDS患者最终进展为急性髓系白血病(Acute myelogenous leukemia,AML)。自然杀伤细胞(Natural killer cells,NK细胞)是淋巴细胞的一种,在正常人外周血淋巴细胞中所占比例约为10%-15%,具有早期可产生细胞因子、趋化因子和非致敏即可溶解靶细胞的能力,因此在机体抗肿瘤、抗病毒免疫中具有重要作用。近年来,人们逐渐发现MDS患者中NK细胞数量和功能均存在异常,并与MDS的病情和疾病进展具有相关性。本研究应用流式细胞术检测MDS患者外周血NK细胞的数量、亚型和受体的表达,应用共培养的方法检测NK细胞对靶细胞的杀伤功能,并与MDS患者的的病情和相关临床指标进行相关性分析。方法收集天津医科大学总医院自2015年10月至2016年6月收治的35例MDS患者及34名正常对照者的外周血标本。第一部分应用流式细胞术检测MDS患者和正常对照者外周血NK细胞(CD3-CD56+)、CD56brightNK细胞(CD3-CD56brightCD16-)、CD56dimNK细胞(CD3-CD56dimCD16+)、T细胞(CD3+)、NKT细胞(CD3+CD56+)的数量,NK细胞的两个亚型即CD56brightNK细胞、CD56dimNK细胞的构成比及CD56brightNK细胞/CD56dimNK细胞;检测NK细胞表面的功能分子NKp30、NKp46、NKG2A的表达情况;检测MDS患者治疗前后NK细胞的数量和功能分子有无变化;根据MDS不同危险度分层进行分析;并与骨髓分类中骨髓原始细胞百分比、外周血中性粒细胞绝对值(ANC)、血红蛋白含量(Hb)做相关性分析。第二部分收集7例MDS患者和8名正常对照者新鲜抗凝外周血,应用免疫磁珠法分选出CD3-CD56+NK细胞,体外应用高浓度IL-2刺激过夜培养后与靶细胞(K562细胞)进行共培养,6h后,收获细胞并标记PI,应用流式细胞术检测NK细胞杀伤功能。结果第一部分(1)NK细胞数量:MDS组NK/Lym%、CD56dimNK/Lym%显著低于对照组,分别为(8.19±5.30 vs 13.81±5.96,P0.001)、(7.95±5.14 vs 12.78±5.74,P=0.001);MDS组CD56brightNK/Lym%高于对照组(0.68±0.33 vs 0.53±0.22,P0.05);MDS组NKT/Lym%、T cells/Lym%与对照组无显著差异,分别为(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细胞亚型,MDS组CD56dimNK/NK%明显低于对照组(91.12±3.49 vs 95.40±2.48,P0.001);CD56brightNK/NK%明显高于对照组(8.87±3.49 vs 4.60±2.48,P0.001);MDS组CD56brightNK/CD56dimNK%细胞明显高于对照组(9.90±4.29 vs4.89±2.77,P0.001);MDS组NK细胞、CD56dimNK、CD56brightNK细胞的绝对值(/ul)均低于对照组,分别为(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细胞表面功能分子的表达:MDS组NK细胞表面NKG2A的表达与对照组无明显差异(37.31±19.60 vs 37.45±14.24,P㧐0.05);而活化性受体NKp30和NKp46的表达明显低于对照组,分别为(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患者治疗后,随着病情好转,NK/Lym%、CD56dimNK/Lym%逐渐恢复,随着病情进展,NK细胞数量进一步下降。(4)在MDS组中,高危组NK/Lym%、CD56dimNK/Lym%明显低于低危组(5.41±0.97 vs 10.13±1.83,P=0.04)、(4.64±0.94 vs 8.81±1.14,P=0.02);而高危组与低危组NKG2A、NKp30、NKp46的表达无明显差异,分别为(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细胞数量与功能分子的表达与临床指标的相关性:MDS患者NK/Lym%、CD56dimNK/Lym%与患者骨髓原始细胞比例呈负相关(r=-0.53和-0.68,P0.05),与外周血血红蛋白含量(Hb)呈正相关(r=0.35和0.50,P0.05);与中性粒细胞绝对值(ANC)呈正相关(r=0.52和0.53,P0.01);NK细胞表面活化性受体NKp46的表达与骨髓原始细胞数呈负相关(r=-0.584,P0.05)。第二部分NK细胞与K562共培养后,MDS组K562细胞的凋亡率低于对照组(7.73±4.0 vs 3.14±2.47,P=0.029)。结论:MDS患者NK细胞数量减少,亚型失衡,活化性受体下降,可能导致其活化不足,杀伤功能下降,从而导致其不能正常行使有效的免疫监视功能,进而不能早期有效清除MDS恶性克隆细胞,导致MDS患者病情进展。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R551.3
本文编号：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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R551.3
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