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

doi:10.12140/j.issn.1000-7423.2022.01.005

中国寄生虫学与寄生虫病杂志 ›› 2022, Vol. 40 ›› Issue (1): 36-42.doi: 10.12140/j.issn.1000-7423.2022.01.005

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

阿卜杜艾尼·啊卜力孜¹^,²(), 排组拉沙拉依阿当¹^,², 塔来提·吐尔干¹^,², 张瑞青¹^,², 王慧²^,³, 张传山²^,³, 邵英梅¹^,², 吐尔干艾力·阿吉¹^,²^,^*()

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

收稿日期:2021-07-26 修回日期:2021-10-23 出版日期:2022-02-28 发布日期:2021-12-28
通讯作者: 吐尔干艾力·阿吉
作者简介:阿卜杜艾尼·啊卜力孜（1986-）,男,博士研究生,从事肝棘球蚴病的诊断、治疗及相关基础研究。E-mail： 531598286@qq.com
基金资助:
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)

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

ABUDUAINI Abulizi¹^,²(), PAIZULA Shalayiadang¹^,², TALAITI Tuergan¹^,², ZHANG Rui-qing¹^,², WANG Hui²^,³, ZHANG Chuan-shan²^,³, SHAO Ying-mei¹^,², TUERGANAILI Aji¹^,²^,^*()

1 Hepatobiliary & Hydatid Disease Department, Digestive & Vascular Surgery Center, First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, China;
2 State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Xinjiang Medical University, Urumqi 830054, China
3 Clinical Medical Institute, First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, China

Received:2021-07-26 Revised:2021-10-23 Online:2022-02-28 Published:2021-12-28
Contact: TUERGANAILI Aji
Supported by:
Opening Foundation of State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia(SKL-HIDCA-2020-24);National Natural Science Foundation of China(8156040098)

摘要/Abstract

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100 μl 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1 μg/ml白细胞介素-12（IL-12）和IL-15各1 μl,Emp组加入1 μg/ml IL-12、IL-15各1 μl和7 081 μg/ml Emp 2.5 μl,转化生长因子-β1（TGF-β1）组（阳性对照组）加入1 μg/ml IL-12、IL-15、TGF-β1各1 μl,不足总量（104.5 μl）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。

关键词: 多房棘球蚴, 虫体蛋白, 自然杀伤细胞, NKG2A

Abstract:

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 100 μl 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-β 1(TGF-β1) group (positive control group). The blank conrol group underwent no further treatment. For the negative control group, 1 μl interleukin-12 (IL-12) and IL-15 (of 1 μg/ml each) were added, while the Emp group was treated with 1 μl IL-12 and IL-15 (at 1 μg/ml each) and 2.5 μl Emp (7 081 μg/ml); to the TGF-β1 group, 1 μl TGF-β1 (1 μg/ml) were added. RPMI 1640 medium was used to adjust the wells to a final volume of 104.5 μl 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 factor α (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-γ was (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-γ was (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⁺NK cells secreting granzyme B, TNF-α, and perforin was insignificant between the Emp griyog and negative control secreting NK cells and NKG2A⁺NK cells (P > 0.05). In the TGF-β1 group, the percentage of NK and NKG2A⁺NK cells secreting IFN-γ was (12.77 ± 2.56)% and (15.17 ± 6.34)%, respectively, which were lower than that in the negative control group (P < 0.01); there was no significant difference in the percentage of NK and NKG2A⁺NK cells secreting granzyme B, TNF-α, and perforin was forund between the Emp and negative control. Of the TGF-β1 group, the percentage of NK cells secreting IFN-γ upon stimulation was lower than that in the Emp group, but the difference was not statistically significant (P > 0.05). Conclusion Emp mediates up-regulation of the expression of NK cell surface receptor NKG2A and inhibits the function of NK cells secretging IFN-γ.

Key words: Echinococcus multilocularis, Protein, Natural Killer cell, NKG2A

中图分类号:

R383.33

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, 王慧, 张传山, 邵英梅, 吐尔干艾力·阿吉. 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[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(1): 36-42.

ABUDUAINI Abulizi, PAIZULA Shalayiadang, TALAITI Tuergan, ZHANG Rui-qing, WANG Hui, ZHANG Chuan-shan, SHAO Ying-mei, TUERGANAILI Aji. Affect of Echinococcus multilocularis protein-mediated NK cell surface receptor NKG2A on the function of NK cells[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(1): 36-42.

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抗体 Antibody	荧光标记 Fluorescent label	克隆号 Clone number
CD3	PE-Cy7	UCHT1
CD56	APC-Cy7	HCD56
CD16	FITC	3G8
NKG2A	PE	131411
NKG2D	PerCP-Cy5.5	1D11
IFN-γ	APC	4S.B3
TNF-α	PE/Dazzle^TM 594	MAb11
Perforin	Brilliant Violet 510^TM	dG9
Granzyme B	Pacific blue	GB11

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

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