胆管癌细胞外泌体对自然杀伤细胞功能影响

Impact of cholangiocarcinoma cell derived exosomes on natural killer cell function

目的探究胆管癌细胞来源的外泌体(RBE-Exos)对自然杀伤(NK)细胞功能的影响及其内在机制。方法采用免疫组化染色对比分析NK细胞在胆管癌组织和癌旁组织中的浸润数量;通过超速离心法从胆管癌细胞中分离出外泌体,透射电镜(TEM)观察RBE-Exos的形态特征,蛋白质印迹实验鉴定RBE-Exos的蛋白标志物;纳米颗粒跟踪分析仪(NTA)检测RBE-Exos粒径分布。NK-92细胞与RBE-Exos共培养后,荧光染色示踪RBE-Exos进入细胞;通过实时荧光定量聚合酶链式反应(qRT-PCR)和蛋白质印迹实验检测黏附分子的表达水平。LDH法检测NK-92细胞的杀伤功能变化。结果免疫组化染色显示,与癌旁组织相比,表达CD56(t=2.977,P=0.008)和NKp46(t=4.530,P<0.001)特异性标志分子的NK细胞在癌组织中的浸润数量减少。RBE-Exos电镜下呈酒窝或杯状,特异性表达CD63和HSP70。NTA结果显示RBE-Exos平均直径为(102.90±7.00)nm。荧光染色显示,培养24 h后RBE-Exos进入NK-92细胞。RBE-Exos处理后,NK-92细胞失去了正常的聚团现象而呈现分散的状态。qRT-PCR结果显示,RBE-Exos处理组黏附分子CD11a(t=4.999,P=0.038)、CD18(t=9.225,P=0.012)和CD54(t=12.050,P=0.007)在转录水平上的表达较PBS组均降低。蛋白质印迹实验结果表明,RBE-Exos处理组较PBS组黏附分子CD11a(t=5.140,P=0.007)、CD18(t=7.177,P=0.002)和CD54(t=8.713,P<0.001)蛋白表达均减少。LDH检测显示,RBE-Exos减弱NK-92细胞对靶细胞的杀伤功能(20∶1,t=3.975,P=0.017;10∶1,t=5.776,P=0.005)。结论胆管癌可能通过释放外泌体抑制NK细胞黏附分子表达从而抑制其在肿瘤部位的浸润。

Objective To investigate the effect of RBE-Exos on the function of natural killer(NK)cells and their underlying mechanisms.Methods Immunohistochemical staining was performed to detect the numbers of NK cells infiltrated into cholangiocarcinoma tissues and paracancerous tissues.Exosomes were isolated from RBE cells by ultracentrifugation.The morphology,size and protein markers of RBE-Exos were idemtified by transmission electron microscopy(TEM),Nanoparticle tracking analysis(NTA)and western blot respectively.Immunofluorescence staining was conducted to show the internalization of RBE-Exos into NK-92 cells.The expression levels of adhesion molecules were analyzed by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and western blot after NK-92 cells were treated with RBEExos.LDH detection was applied to analyze the killing activity of NK-92 cells.Results The results of immunohistochemical staining showed that the numbers of CD56(t=2.977,P=0.008)or NKp46(t=4.530,P<0.001)positive NK cells in tumor tissues were lower than those in paracancerous tissues.The morphology of isolated RBE-Exos was identified by TEM as typical dimpled and cup-shaped particles with expression of CD63 and HSP70.NT A detection showed that the average diameter of RBE-Exos was(102.90±7.00)nm.After 24 hours of incubation,RBE-Exos was observed to enter NK-92 cells by fluorescent staining.NK-92 cells lost the normal character of clustered growing and presented in a dispersed status after treated with RBE-Exos.The results of qRT-PCR indicated that the expression of adhesion molecules CD11a(t=4.999,P=0.038),CD18(t=9.225,P=0.012),and CD54(t=12.050,P=0.007)at the transcriptional levels were reduced in the RBE-Exos-treated NK-92 cells compared with the PBS group.Western blot results displayed that the protein expression of adhesion molecules CD11a(t=5.140,P=0.007),CD18(t=7.177,P=0.002),and CD54(t=8.713,P<0.001)were all decreased in the RBE-Exos-treated NK-92 cells compared with the PBS group.LDH detection showed that the killing activity of NK-92 cells against target cells was attenuated by RBE-Exos(20:1,t=3.975,P=0.017;10:1,t=5.776,P=0.005).Conclusions Cholangiocarcinoma cells may inhibit the expression of adhesion molecules in NK cells by releasing exosomes,consequently inhibiting NK cells infiltration into tumor site.