NKG2D/RAE-1通路在介导NK/T细胞杀伤垂体瘤细胞中的作用及意义

Effect and significance of NKG2D/RAE-1 pathway in the process of NK/T cells killing hypophysoma cells

目的观察NKG2D/RAE-1通路在介导自然杀伤(NK)细胞、CD8^+T细胞(两种细胞简称NK/T细胞)杀伤垂体瘤细胞中的作用,从细胞学水平探讨垂体瘤的免疫逃逸机制。方法以小鼠垂体瘤细胞系At T20为研究对象,采用免疫荧光标记法检测NKG2D配体RAE-1在垂体瘤细胞的表达水平及定位分布情况。采用NK/T细胞分选试剂盒分离NK/T细胞,将NK/T细胞放入Transwell小室的上室,靶细胞At T20置入Transwell小室的下室,随机分为对照组、实验组。对照组在常规条件下共培养0、4、8、24 h,实验组在培养液中加入阻断RAE-1的抗体(AntiRAE1抗体)共培养0、4、8、24 h;采用MTT法检测两组各时间点细胞增殖抑制率。结果 RAE-1在At T20细胞膜明显表达,在细胞质内亦有弱表达。在At T20细胞中RAE-1蛋白阳性表达率为100%,其中强表达率为30%、中表达率为60%、弱表达率为10%,无阴性表达。对照组培养4、8、24 h时细胞增殖抑制率明显高于0 h时(P均<0.05);实验组培养0、4、8、24 h时增殖抑制率逐渐升高(P均<0.05),培养8、24 h时增殖抑制率均明显高于同时间点对照组(P均<0.05)。结论 NK/T细胞通过NKG2D/RAE-1通路杀灭垂体瘤细胞,RAE-1可负反馈作用于NK/T细胞,从而抑制免疫细胞对肿瘤细胞的杀伤活性。NKG2D/RAE-1通路可能作为垂体瘤的免疫治疗靶点。

Objective To observe the effect of NKG2 D/RAE-1 pathway on mediating natural killer（ NK） cell and CD8^＋T cell（ both cells were referred to as NK/T cells） killing hypophysoma cells and to investigate the immune escape mechanism of hypophysoma from the cytological level. Methods The hypophysoma cell line At T20 was selected. The immunofluorescent labeling method was used to detect the expression level of NKG2 D ligand RAE-1,its position and distribution in the hypophysoma cells. NK/T cells were isolated by NK/T cell sorting kit,and NK/T cells were placed in the upper chamber of the Transwell chamber,the target cells At T20 were placed in the lower chamber of the Transwell chamber and then were randomly divided into the control group and experimental group. The control group was co-cultured under normal conditions for 0,4,8,and 24 h. In the experimental group,the anti-RAE1 antibody was added to the culture medium,and the cells were co-cultured for 0,4,8,and 24 h. MTT assay was used to detect the inhibitory rate of cell proliferation at each time point. Results RAE-1 was clearly expressed in At T20 cell membrane,and there was a weak expression in the cytoplasm. The positive expression rate of RAE-1 protein in At T20 cells was 100%,among which the strong expression rate was 30%（ 15/50）,the moderate expression rate was 60%,the weak expression rate was 10%,and there was no negative expression. In the control group,at 4,8 and 24 h after culture,NK/T cell proliferation inhibition rate increased as compared with that at 0 h（ all P〈0. 05）. In the experimental group,the proliferation inhibition rate gradually increased at0,4,8 and 24 h（ all P〈0. 05）,and at 8 and 24 h,the proliferation inhibition rates in the experimental group were significantly higher than those of the control group（ all P〈0. 05）. Conclusions NK/T cells kill the hypophysoma cells through NKG2 D/RAE-1 pathway,meanwhile,RAE-1 has a negative feedback effect on NK/T cells and consequently inhibits the killing activity of immune cells against the tumor cells. NKG2 D/RAE1 pathway can be used as immunotherapy target for hypophysoma.