食管鳞癌细胞培养上清液对正常CD3^+CD56^+NKT细胞亚型、表面受体及效应分子表达的影响

Effects of culture supernatant of esophageal squamous-cell carcinoma cells on the subtype,surface receptor and effector molecule of normal CD3^+CD56^+NKT cells

目的观察食管鳞癌细胞株培养上清液对健康人外周血CD3^+CD56^+NKT细胞亚型、表面受体及效应分子表达的影响。方法取30例健康志愿者的外周血,用淋巴细胞分离液常规方法分离外周血单核细胞。收集食管鳞癌细胞株Eca9706、Kyse150的培养上清液,将上清液与含10%胎牛血清的RPMI1640培养基按1∶1混合以配制条件培养基。将外周血单核细胞分为对照组、Eca9706组、Kyse150组。对照组细胞仅用含IL-2、10%胎牛血清的RPMI1640培养液培养;Eca9706组、Kyse150组细胞加入IL-2刺激24 h后,分别更换条件培养基(Eca9706细胞培养上清液、Kyse150细胞培养上清液)培养48 h。收集各组细胞,选择流式细胞仪的CD3^+CD56^+的细胞群设计分析,分析各组CD3^+CD56^+NKT细胞亚型,检测CD3^+CD56^+NKT细胞中的表面受体(NKG2D、NKG2A、NKP30、NKP44、CD158b、CD271)及效应分子(穿孔素、颗粒酶、Ki-67)。结果 Eca9706组及Kyse150组的CD3^+CD56^+NKT细胞中CD4^+CD8-NKT细胞比例高于对照组,CD4-CD8-NKT细胞比例低于对照组(P均<0.05)。Eca9706组、Kyse150组的CD3^+CD56^+NKT细胞中NKG2A、CD158b表达率高于对照组,NKP44、NKP30、CD271表达率低于对照组(P均<0.05)。Eca9706组的CD3^+CD56^+NKT细胞中穿孔素表达率低于对照组(P<0.05);Eca9706组的CD3^+CD56^+NKT细胞中Ki-67表达率高于对照组(P<0.05)。结论食管鳞癌细胞株培养上清液能改变正常CD3^+CD56^+NKT细胞的亚型,使细胞表面活化型受体和抑制型受体表达比例失衡,并下调效应分子穿孔素的表达,使CD3^+CD56^+NKT细胞的免疫监视功能受损。上述变化可能是食管鳞癌细胞逃避机体免疫监视的机制之一。

Objective To observe the effects of culture supernatant of esophageal squamous-cell carcinoma（ ESCC）on the subtype,surface receptor,and effector molecule of CD3~＋CD56~＋NKT cells in the healthy volunteers. Methods Peripheral blood from 30 healthy volunteers was isolated to get monocytes by routine method of lymphocyte separation solution. The culture supernatant of ESCC Eca9706 and Kyse150 cells was collected,and the supernatant was mixed with RPMI1640 medium containing 10% fetal bovine serum by the proportion of 1： 1 to prepare the conditional medium. Peripheral monocytes were divided into the control group,Eca9706 group,and Kyse150 group. The cells of the control group were cultured with RPMI1640 medium containing IL-2 and 10% fetal bovine serum. After the cells of Eca9706 group and Kyse150 group were stimulated by IL-2 for 24 h,we replaced the conditioned medium with Eca9706 cell culture supernatant and Kyse150 cell culture supernatant,respectively,and continued to culture for 48 h. We collected the cells of each group and chose the CD3~＋CD56~＋cells for analysis. We analyzed the CD3~＋CD56~＋NKT cell subsets in each group and detected the surface receptors（ NKG2 D,NKG2 A,NKP30,NKP44,CD158 b,CD271） and effector molecule（ perforin,granular enzyme,and Ki-67） in the CD3~＋CD56~＋NKT cells. Results The proportion of CD4~＋CD8-NKT cells in the CD3~＋CD56~＋NKT cells of the Eca9706 group and Kyse150 group were both higher than that in the control group,and the proportion of CD4-CD8-NKT cells was lower than that in the control group（ all P〈0. 05）. The expression rates of NKG2 A and CD158 b in the CD3~＋CD56~＋NKT cells of the Eca9706 group and Kyse150 group were higher than those of the control group,and the expression rates of NKP44,NKP30 and CD271 were lower than those of the control group（ all P〈0. 05）.The expression rate of perforin in the CD3~＋CD56~＋NKT cells of the Eca9706 group was lower than that in the control group（ P〈0. 05）. The expression rate of Ki-67 in the CD3~＋CD56~＋NKT cells of the Eca9706 group was higher than that of the control group（ P〈0. 05）. Conclusions The ESCC culture supernatant can change the subtypes of CD3~＋CD56~＋NKT cells,lose the balance between activated receptor and inhibitory receptor of cell surface,down-regulate the expression of perforin,and impair the immune surveillance function of CD3~＋CD56~＋NKT cells. These changes may be one of the mechanisms by which ESCC cells escape the immune surveillance.