食管鳞癌细胞对自然杀伤细胞的生物学效应的影响

Effect of esophageal squamous cell carcinoma cells on the biological activity of natural killer cells

摘要:

目的：研究食管鳞癌原代细胞对自然杀伤细胞表型及功能的调控。方法：收集新鲜食管鳞癌组织，采用改良组织块结合酶消化法在体外培养原代食管鳞癌细胞，通过裸鼠皮下成瘤模型及免疫组织化学技术鉴定食管鳞癌细胞；收集正常人外周血单个核细胞，磁珠法负性分离NK细胞；将原代食管鳞癌细胞培养上清与NK细胞共培养（根据共培养条件分组为NC-NK，ESCC1-NK，ESCC2-NK），使用流式细胞术检测NK细胞表面及胞内分子的表达；通过裸鼠皮下成瘤及肺转移模型研究原代食管鳞癌细胞对NK细胞肿瘤杀伤作用的影响。结果：与NC-NK组比较，ESCC1-NK组、ESCC2-NK组活化型受体NKG2D（56.84±1.28，41.89±2.17，29.40±1.32）（P=0.008，P=0.001）、NKP30（45.79±1.90，19.54±1.27，26.59±1.47）（P=0.001，P=0.003）及CD16（84.82±1.38，55.95±2.29，36.07±1.97）（P=0.001，P=0.000）表达降低；NK细胞胞内效应分子颗粒酶B（94.87±1.04，80.52±0.99，78.67±2.73）（P=0.013，P=0.008）及增殖相关抗原Ki67（70.15±2.35，52.31±1.74，50.02±2.68）（P=0.017，P=0.011）表达下降；活化型受体NKP44、NKP46、CD226，穿孔素及抑制型受体NKG2A、CD158b的表达差异无统计学意义。动物实验显示：皮下瘤体体积ESCC1-NK组和NC-NK组组间有差异（F=54.689，P=0.000）；ESCC1-NK处理组肺转移灶数（41.00±3.11）多于NC-NK处理组（25.25±2.32）（t=4.068，P=0.007）。结论：原代食管鳞癌细胞可能通过抑制NK细胞活化及增殖、下调NK细胞杀伤效应分子的表达来抑制NK细胞肿瘤杀伤效应，从而逃逸机体免疫监视。

Abstract:

Objective：To study the regulation and control of the phenotype and function of natural killer（NK） cells by the primary cells of esophageal squamous cell carcinoma（ESCC）. Methods：Fresh ESCC tissue was collected，from which primary ESCC cells were cultured in vitro by the modified tissue block enzymolytic method，and identified using a subcutaneous tumor-formation model in nude mice and the immunohistochemical technique. Human peripheral blood mononuclear cells were collected from healthy subjects，from which the NK cells were isolated by negative-selection magnetic beads，and co-cultured with the supernatant of the primary ESCC cells obtained above. The cultured cells were divided into groups of NC-NK，ESCC1-NK，and ESCC2-NK according to their respective culture conditions. Flow cytometry was used to determine the molecular expression both on the surface of and within the NK cells. The subcutaneous tumor-formation model and lung metastasis model were used to study the effect of primary ESCC cells on the killing effect of NK cells in nude mice. Results：Compared with the NC-NK group，the ESCC1-NK group and the ESCC2-NK group had significantly reduced expression of the activated receptors NKG2D（56.84±1.28 vs. 41.89±2.17 and 29.40±1.32；P=0.008 and 0.001，respectively），NKP30（45.79±1.90 vs. 19.54±1.27 and 26.59±1.47；P=0.001 and 0.003，respectively），and CD16（84.82±1.38 vs. 55.95±2.29 and 36.07±1.97；P=0.001 and 0.000，respectively） as well as significantly reduced expression of the effector molecule granzyme B（94.87±1.04 vs. 80.52±0.99 and 78.67±2.73；P=0.013 and 0.008，respectively） and the prolif－eration-associated antigen Ki67（70.15±2.35 vs. 52.31±1.74 and 50.02±2.68；P=0.017 and 0.011，respectively） in NK cells. There were no significant differences between the three groups in the expression of the activated receptors NKP46，CD226，and NKP44 as well as perforin and the inhibitory receptors NKG2A and CD158b. Animal experiments showed that there was a significant difference between the ESCC1-NK group and the NC-NK group in the volume of subcutaneous tumors（F=54.689，P=0.000） with a significantly greater number of lung metastases observed in the ESCC1-NK group（41.00±3.11 vs. 25.25±2.32，t=4.068，P=0.007）. Conclusion：Primary ESCC cells may suppress the tumor-killing effect of NK cells by inhibiting the activation and proliferation of NK cells and down-regulating the expression of the effector molecules in NK cells，thereby escaping the immune surveillance in the body.