多房棘球蚴虫体蛋白介导NK细胞表面受体NKG2A对NK细胞功能的影响  

Affect of Echinococcus multilocularis protein-mediated NK cell surface receptor NKG2A on the function of NK cells

作　　者：阿卜杜艾尼·啊卜力孜 排组拉沙拉依阿当 塔来提·吐尔干 张瑞青[1,2] 王慧[2,3] 张传山[2,3] 邵英梅 吐尔干艾力·阿吉 ABUDUAINI Abulizi;PAIZULA Shalayiadang;TALAITI Tuergan;ZHANG Rui-qing;WANG Hui;ZHANG Chuan-shan;SHAO Ying-mei;TUERGANAILI Aji(Hepatobiliary&Hydatid Disease Department,Digestive&Vascular Surgery Center,First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China;State Key Laboratory of Pathogenesis,Prevention and Treatment of High Incidence Diseases in Central Asia,Xinjiang Medical University,Urumqi 830054,China;Clinical Medical Institute,First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China)

机构地区：[1]新疆医科大学第一附属医院消化血管中心肝胆包虫病外科,乌鲁木齐830054 [2]新疆医科大学省部共建中亚高发病成因与防治国家重点实验室,乌鲁木齐830054 [3]新疆医科大学第一附属医院临床医学研究院,乌鲁木齐830054

出　　处：《中国寄生虫学与寄生虫病杂志》2022年第1期36-42,共7页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：省部共建中亚高发病成因与防治国家重点实验室开放课题(SKL-HIDCA-2020-24);国家自然科学基金(8156040098)。

摘　　要：目的探讨多房棘球蚴虫体蛋白(Emp)介导自然杀伤(NK)细胞表面抑制性受体NK细胞凝集素样受体亚家族C成员A (NKG2A)对NK细胞功能的影响。方法采集健康志愿者外周血,纯化NK细胞,按0.3×10^(6)个/孔(重悬至100μl RPMI 1640培养基),加至96孔细胞培养板,空白对照组不作处理,阴性对照组加入1μg/ml白细胞介素-12 (IL-12)和IL-15各1μl,Emp组加入1μg/ml IL-12、IL-15各1μl和7 081μg/ml Emp 2.5μl,转化生长因子-β1 (TGF-β1)组(阳性对照组)加入1μg/ml IL-12、IL-15、TGF-β1各1μl,不足总量(104.5μl)的用RPMI 1640培养基调整,分别体外刺激培养24 h后,利用流式细胞术检测NK细胞表面受体NKG2A表达情况以及NK细胞和NKG2A^(+)NK细胞分泌细胞因子[γ干扰素(IFN-γ)、颗粒酶B、肿瘤坏死因子-α(TNF-α)、穿孔素]的功能变化。采用单因素方差分析法进行差异性分析,LSD或Dunnett检验法分析组间差异。结果 Emp组和TGF-β1组表达NKG2A的NK细胞百分比分别为(3.40±1.53)%、(3.00±1.07)%,均高于阴性对照组的(0.70±0.56)%(P<0.01)。Emp组分泌IFN-γ的NK细胞百分比为(42.38±15.94)%,与阴性对照组的(61.18±7.18)%比较差异无统计学意义(P>0.05);Emp组分泌IFN-γ的NKG2A^(+)NK细胞百分比为(25.25±11.57)%,低于阴性对照组的(48.88±12.78)%(P<0.05);两组分泌颗粒酶B、TNF-α、穿孔素的NK细胞和NKG2A^(+)NK细胞百分比差异均无统计学意义(P>0.05)。TGF-β1组分泌IFN-γ的NK细胞和NKG2A^(+)NK细胞百分比分别为(12.77±2.56)%、(15.17±6.34)%,均低于对应阴性对照组(P<0.01);两组分泌颗粒酶B、TNF-α、穿孔素的NK细胞和NKG2A^(+)NK细胞百分比之间差异均无统计学意义(P>0.05)。TGF-β1组NK细胞经TGF-β1刺激后分泌的IFN-γ百分比低于Emp组,但两者差异无统计学意义(P>0.05)。结论 Emp通过介导NK细胞表面受体NKG2A的表达上调而抑制NK细胞分泌IFN-γ的功能。Objective To investigate the affect of Echinococcus multilocularis protein mediated natural killer(NK) cell surface inhibitory receptor NK cell lectin-like receptor subfamily C member A (NKG2A) on the function of NK cells.Methods Peripheral blood samples were colleted from the participants for NK cell purification.An aliquate of 0.3×10^(6)NK cells were resuspended in 100μl RMPI 1640 medium,which was transferred into a 96-well plate.Four test groups were assigned,including blank control,negative control,E.multilocularia protein (Emp)group,and transforming growth factor-β1 (TGF-β1) group (positive control group).The blank conrol group underwent no further treatment.For the negative control group,1μl interleukin-12 (IL-12) and IL-15 (of 1μg/ml each) were added,while the Emp group was treated with 1μl IL-12 and IL-15 (at 1μg/ml each) and 2.5μl Emp (7 081μg/ml);to the TGF-β1 group,1μl TGF-β1 (1μg/ml) were added.RPMI 1640 medium was used to adjust the wells to a final volume of 104.5μl when appropriate.Flow cytometry analysis was used to quantify the expression of NKG2A on NK cells and functional changes of NK cells and NKG2A^(+)NK in secretion of cytokines[interferon-γ(IFN-γ),granzyme B,tumor necrosis factorα(TNF-α) and perforin]after culture stimulated for 24 hours in vitro.The data were analyzed using One-way ANOVA for difference analysis,and LSD or Dunnett test for comparison of the difference between groups.Results The percentage of NKG2A^(+)NK cells in Emp group and TGF-β1 group were (3.40±1.53)%and (3.00±1.07)%,respectively,which were significantly higher than that in the negative control group (0.70±0.56)%(P<0.01).In the Emp group,the percentage of NK cells secreting IFN-γwas (42.38±15.94)%,having was no significant difference compared to the negative control group (61.18±7.18)%(P>0.05).The percentage of NKG2A^(+)NK cells secreting IFN-γwas (25.25±11.57)%,which was lower than that in the negative control group (48.88±12.78)%(P<0.05);the difference in the percentage of NK and NKG2A^(

关 键 词：多房棘球蚴 虫体蛋白 自然杀伤细胞 NKG2A 

分 类 号：R383.33[医药卫生—医学寄生虫学]