舒尼替尼联合NK细胞对肾透明细胞癌细胞株的协同杀伤作用及其机制

doi:10.13481/j.1671-587x.20150414

吉林大学学报(医学版) ›› 2015, Vol. 41 ›› Issue (04): 742-746.doi: 10.13481/j.1671-587x.20150414

舒尼替尼联合NK细胞对肾透明细胞癌细胞株的协同杀伤作用及其机制

张辉见¹, 党强¹, 殷海霞¹, 彭佩丹¹, 刘成山¹, 黄宇贤²

1. 南方医科大学南方医院泌尿外科, 广东广州 510515;
2. 南方医科大学珠江医院血液科, 广东广州 510282

收稿日期:2015-05-13 发布日期:2015-08-01
通讯作者: 黄宇贤,副主任医师(Tel:020-62782322,E-mail:hyx6610@163.com) E-mail:hyx6610@163.com
作者简介:张辉见(1977-),男,广东省揭阳市人,主治医生,在读医学硕士,主要从事泌尿系统肿瘤综合治疗方面的研究。
基金资助:
国家自然科学基金资助课题(81302372)

Synergistic effect of sunitinib combined with NK cells on renal clear cell carcinoma cell lines and its mechanism

ZHANG Huijian¹, DANG Qiang¹, YIN Haixia¹, PENG Peidan¹, LIU Chengshan¹, HUANG Yuxian²

1. Department of Urinary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China;
2. Department of Hematology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China

Received:2015-05-13 Published:2015-08-01

摘要/Abstract

摘要：

目的: 探讨舒尼替尼联合NK细胞对肾透明细胞癌786-0细胞的协同杀伤作用,并阐明其机制。方法: 常规体外培养786-0细胞,分为未处理组和舒尼替尼组,同时设阳性对照组(K562细胞);免疫磁珠技术分离人外周血NK细胞。MTT法检测舒尼替尼对786-0细胞的药物敏感性,流式细胞术检测NK细胞纯度及经舒尼替尼处理前后786-0细胞NKG2D配体(NKG2DLs)表达率,LDH释放测定法检测NK细胞对未处理组、阳性对照组和舒尼替尼组786-0细胞的杀伤活性。结果: 786-0细胞对舒尼替尼的半数抑制浓度(IC₅₀)为(4.45±0.65)μmol·L^-1。分选后NK细胞中CD3^-CD16⁺CD56⁺细胞的纯度达72%以上;经舒尼替尼处理后靶细胞NKG2DLs中MICA、MICB和ULBP2 表达率明显高于处理前(P<0.05)。当效靶比为10:1和20:1时,各组靶细胞的杀伤活性比较差异均有统计学意义 (F=166.18, P＝0.000;F=52.87, P＝0.000),且NK细胞对舒尼替尼组786-0细胞的杀伤活性明显高于未处理组(P<0.05)。结论: 舒尼替尼使肿瘤细胞对NK细胞的杀伤敏感性增强,其协同作用可能与舒尼替尼选择性诱导肿瘤细胞高表达NKG2DLs(MICA、MICB和ULBP2)有关。

关键词: 舒尼替尼, 自然杀伤细胞, 肾透明细胞癌, 自然杀伤细胞2族成员D

Abstract:

Objective To investigate the synergistic effect of sunitinib combined with NK cells on the renal clear cell carcinoma 786-0 cells,and to clarify the mechanism. Methods The 786-0 cells were cultivated by routine method in vitro and divided into untreated group and sunitinib group,meanwhile positive control group (K562 cells) was set up.The human NK cells were isolated by magnetic activated cell sorting (MACS).The sensitivity of 786-0 cells to sunitinib was analyzed by MTT assay.Flow cytometry was used to evaluate the purity of isolated cells and the expression rates of NKG2D-ligands (NKG2DLs) on the target cells before and after treatment of sunitinib.Subsequently,the cytotoxic sensitivities of 786-0 cells to NK cells in untreated group,positive control group and sunitinib group were measured by LDH releasing assay. Results The IC₅₀ values of sunitinib for 786-0 cells was (4.45±0.65) μmol·L^-1.More than 72% of isolated NK cells showed to be CD3^-CD16⁺CD56⁺ cells which would definitely meet the needs of experiments. The expressions rates of MICA,MICB,ULBP2 on the target cells incubated with sunitinib were respectively increased from (3.52±0.29)%,(3.10±0.73)%,(5.12±4.77)% to (15.45±2.14)%,(21.95±3.08)%,(51.71±5.33)%.Compared with untreated group,the expression rates of MICA,MICB and ULBP2 in treatment group were significantly increased(P<0.05).At the E:T ratio of 10:1 and 20:1,the cytotoxic sensitivities of 786-0 cells to NK cells were increased from (9.71±0.88)% and (21.28±0.32)% in untreated group to (20.83±1.28)% and (35.11±0.70)% in sunitinib group(F＝166.18,P＝0.000;F=52.87,P=0.000);the cytotoxic sensitivities of NK cells to the 786-0 cells in sunitinib group were higher than that in untreated group (P<0.05). Conclusion Sunitinib can selectively regulate the expressions of NKG2DLs (MICA/B and ULBP2) in the 786-0 cells,which can result in higher cytotoxic sensitivity to NK cells.Sunitinib combined with NK cells has synergistic effect on the renal clear cell carcinoma cells.

Key words: sunitinib, natural killer cells, renal clear cell carcinoma, natural killer group 2 member D

中图分类号:

R737.11

张辉见, 党强, 殷海霞, 彭佩丹, 刘成山, 黄宇贤. 舒尼替尼联合NK细胞对肾透明细胞癌细胞株的协同杀伤作用及其机制[J]. 吉林大学学报(医学版), 2015, 41(04): 742-746.

ZHANG Huijian, DANG Qiang, YIN Haixia, PENG Peidan, LIU Chengshan, HUANG Yuxian. Synergistic effect of sunitinib combined with NK cells on renal clear cell carcinoma cell lines and its mechanism[J]. Journal of Jilin University Medicine Edition, 2015, 41(04): 742-746.

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舒尼替尼联合NK细胞对肾透明细胞癌细胞株的协同杀伤作用及其机制

Synergistic effect of sunitinib combined with NK cells on renal clear cell carcinoma cell lines and its mechanism

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