食管鳞癌上皮细胞间质转化对NK细胞活化及其生物学作用的研究

Effect of Epithelial-to-mesenchymal Transition on Biological Activity of NK Cells in Esophageal Squamous Cell Carcinoma

目的探讨食管鳞癌上皮细胞间质化(EMT)对自然杀伤(NK)细胞生物学活性的影响。方法通过Western blot检测EMT化相关蛋白E-cadherin和Vimentin表达,从6株细胞系中筛选出EMT化和非EMT化食管鳞癌细胞株;收集10例正常人外周血,负性分选NK细胞;根据共培养不同的条件培养基分为RPMI 1640正常培养基刺激-NK组(NC组)、EC9706细胞上清液刺激-NK组(EC9706组)与KYSE150细胞上清液刺激-NK组(KYSE150组),将不同的条件培养基加入NK细胞共培养72h,流式细胞术检测NK细胞表面/胞内分子的表达。10ng/mL转化生长因子-β(TGF-β)处理KYSE150细胞株7d诱导EMT化后,收集KYSE150EMT和KYSE150上清液,分别与NK细胞共培养,72h后以流式细胞术检测NK细胞表面/胞内分子的表达、CD107a脱颗粒能力、靶细胞K562杀伤效应、NK细胞增殖及凋亡。结果 6株细胞系中筛选出2株EMT化和4株非EMT化的食管鳞癌细胞株,从中各选出一株(EC9706和KYSE150)用于后续实验;NK细胞负性筛选达到90%以上的纯度,T细胞<1%。不同的食管鳞癌细胞上清液刺激后,3组NK细胞表面活化型、抑制型受体,效应分子结果显示:与NC组和KYSE150组比较,EC9706组活化型受体NKP30、NKG2D、NKP44表达,颗粒酶释放均降低(P<0.05);抑制型受体NKG2A和CD158b表达增加(P<0.05);NKP46、CD226、CD16表达和穿孔素释放在3组间差异无统计学意义。与KYSE150上清刺激比较,KYSE150EMT上清刺激后,NK细胞活化型受体NKP30、NKG2D、NKP44表达,穿孔素释放均降低;CD107a脱颗粒作用、靶细胞K562杀伤效应均减弱,NK细胞Ki67增殖指数下降(P<0.05);抑制型受体NKG2A和CD158b表达增加(P<0.05);NKP46、CD226、CD16表达,颗粒酶释放及NK细胞凋亡在两组间差异无统计学意义。结论食管鳞癌细胞EMT化可能通过抑制NK细胞活化、减少NK细胞杀伤效应分子的分泌来降低NK细胞杀伤效应,从机体免疫监视中逃逸。

Objective To investigate the effect of epithelial-mesenchymal transition(EMT)on biological activity of natural killer(NK)cells in esophageal squamous cell carcinoma(ESCC).Methods Western blot selected EMT EC9706 and non EMT KYSE150 from six esophageal cancer cell lines.NK cells were collected from 10 cases of healthy volunteers.According to different co-culture conditions,the medium was divided into 1640 normal medium stimulus-NK group(NC group),EC9706 cell supernatant stimulus-NK group(EC9706 group)and KYSE150 cell supernatant stimulus-NK group(KYSE150group).The expression of NK cells surface/intracellular molecules was detected by flow cytometry after co-culture of NK cells with different conditioned medium for 72 h.10ng/mL transforming growth factor-β(TGF-β)treated KYSE150 cell line for 7dto induce EMT.KYSE150 EMT and KYSE150 supernatant were collected and co-cultured with NK cells,respectively.72 hlater,flow cytometry was used to detect the expression of surface/intracellular molecules of NK cells,degranulation ability of CD107 a,killing effect of target cell K562,proliferation and apoptosis of NK cells.Results Two EMT-treated and four nonEMT-treated esophageal squamous cell carcinoma lines were selected from the six strains,and one EC9706 and one KYSE150 respectively were selected for subsequent experiments.The purity of NK cells was more than 90%and T cells<1%.After the supernatant of esophageal squamous cell carcinoma cells was stimulated,the surface activation type and inhibitory type receptors of NK cells in the three groups were stimulated.The effector molecule results showed that:compared with NC group and KYSE150 group,the expressions of activated type receptors NKP30,NKG2 Dand NKP44in EC9706 group were decreased,and the release of granulozyme was decreased(P<0.05).Expressions of inhibitory receptor NKG2 Aand CD158bincreased(P<0.05).NKP46,CD226,CD16 expressions and perforin release showed no statistically significant difference among the three groups.Compared with KYSE150 supernatant stimulation,the expressions of activated receptors NKP30,NKG2 D and NKP44 decreased after KYSE150 EMT supernatant stimulation,and perforin release decreased.The degranulation of CD107 aand the killing effect of target cell K562 were decreased,and the proliferation index of NK cell Ki67 was decreased(P <0.05).Expressions of inhibitory receptor NKG2 A and CD158bincreased(P <0.05).The expressions of NKP46,CD226 and CD16,granulozyme release and apoptosis of NK cells were not statistically significant.Conclusion EMT of esophageal cancer cells can escape the immune surveillance by inhibit the activity of NK cells and reduce the release of effective molecules.