摘要
目的:探究杀伤细胞免疫球蛋白样受体2DL4(killer cell Ig-like receptor 2DL4,KIR2DL4)在急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)患者外周血中的表达,及其对自然杀伤细胞(nature killer,NK)杀伤功能的影响。方法:从ALL患者及健康志愿者的外周血中分离出单个核细胞,采用实时荧光定量PCR检测单个核细胞中KIR2DL4的表达差异;从单个核细胞中分选NK细胞,通过流式细胞仪检测CD3-CD56+标记的NK细胞比例,并分析NK细胞表面KIR2DL4与Molt-4细胞表面人白细胞抗原G(human leukocyte antigen G,HLA-G)的表达水平。分别以NK细胞、KIR2DL4阻断抗体作用的NK细胞、IgG1抗体作用的NK细胞作效应细胞,Molt-4细胞为靶细胞,在不同效靶比(5:1、10:1、20:1)下,利用ELISA检测细胞上清液中干扰素(interferon-gamma,IFN-γ)与肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平;乳酸脱氢酶(lactate dehydrogenase,LDH)释放法检测不同处理的NK细胞对Molt-4细胞的杀伤作用;并在20∶1的效靶比下,通过激光共聚焦显微镜观察各处理下Molt-4细胞形态学改变,流式细胞术检测不同处理的NK细胞脱颗粒情况。结果:ALL患者外周血中KIR2DL4 mRNA相对表达量显著高于健康志愿者(P<0.05);分选后CD3-CD56+标记的NK细胞纯度达到90%以上;与NK细胞和Molt-4细胞共培养相比,阻断KIR2DL4能够提高细胞上清液中NK细胞分泌因子IFN-γ与TNF-α水平(P<0.05),提高NK细胞对Molt-4细胞的杀伤率(P<0.05),使Molt-4细胞形变与核固缩更明显,并提高NK细胞中CD107α表达水平(P<0.05)。结论:KIR2DL4在ALL患者外周血中高表达,而阻断KIR2DL4能够促进NK细胞分泌IFN-γ、TNF-α及脱颗粒,提高其对Molt-4细胞的杀伤功能。
Objective:To investigate the expression of killer cell Ig-like receptor 2DL4(KIR2DL4)in peripheral blood of patients with acute lymphoblastic leukemia(ALL),and its effect on nature killer(NK)cells of the killing function.Methods:Mononuclear cells were isolated from the peripheral blood of ALL patients and healthy volunteers,and real-time fluorescence quantitative PCR was used to detect the difference in the expression of KIR2DL4 in mononuclear cells.NK cells were sorted from mononuclear cells.The proportion of CD3-CD56+labeled NK cells was detected by flow cytometry,and the expression levels of KIR2DL4 on NK cells and human leukocyte antigen G(HLA-G)on Molt-4 cells were analyzed.The levels of interferon-gamma(IFN-γ)and tumor necrosis factor-α(TNF-α)in cell supernatants were measured by ELISA at different potency-to-target ratios(5∶1,10∶1,20∶1)using NK cells,NK cells after the action of KIR2DL4 blocking antibody,NK cells after the action of IgG1 antibody as effector cells,and Molt-4 cells as target cells,respectively.Lactate dehydrogenase(LDH)release assay was used to detect the killing effect of NK cells with different treatments on Molt-4 cells.At the effect-target ratio of 20∶1,the morphological changes of Molt-4 cells under each treatment were observed by laser confocal microscopy,and the de-granulation of NK cells in different treatments was detected by flow cytometry.Results:The relative expression of KIR2DL4 mRNA in peripheral blood of ALL patients was significantly higher than that of healthy volunteers(P<0.05).The purity of CD3-CD56+labeled NK cells after sorting reached was more than 90%.Compared with the co-culture of NK cells and Molt-4 cells,blocking KIR2DL4 increased the levels of NK cell-secreted factors IFN-γand TNF-αin the cell supernatant(P<0.05),and the killing rate of NK cells to Molt-4 cells was increased(P<0.05).The deformation and nuclear pyknosis of Molt-4 cells were more obvious,and the expression level of CD107αin NK cells was increased(P<0.05).Conclusion:KIR2DL4 was highly expressed in the peripheral blood of ALL patients,and blocking KIR2DL4 could promote the secretion of IFN-γ,TNF-αand degranulation by NK cells,and improve the killing function of Molt-4 cells.
作者
阿迪娜·乌提库尔
石雨薇
Adina·Wutikuer;SHI Yuwei(Department of Hematology,the Second Affiliated Hospital of Xinjiang Medical University,Xinjiang Urumqi 830000,China)
出处
《现代肿瘤医学》
CAS
北大核心
2023年第3期450-455,共6页
Journal of Modern Oncology
基金
新疆维吾尔自治区自然科学基金(编号:2019D01C315)。
