CD47对鼻咽癌干细胞影响及机制研究  

Effect of CD47 on nasopharyngeal carcinoma stem cell and its mechanism

作　　者：于肖肖 赵云腾[1] 张梦雯 唐浩程 韩日[1] 王剑祁 王显文 汪珂 李刚[1] YU Xiao-xiao;ZHAO Yun-teng;ZHANG Meng-wen;TANG Hao-cheng;HAN Ri;WANG Jian-qi;WANG Xian-wen;WANG Ke;LI Gang(Department of Otorhinolaryngology,Nanfang Hospital,Southern Medical University,Guangzhou510515,P.R.China;Department of Otorhinolaryngology,Central Hospital,Jiangmen,Guangdong529000,P.R.China;Department of Otorhinolaryngology,Third Affiliated Hospital of Southern Medical University,Guangzhou510515,P.R.China)

机构地区：[1]南方医科大学南方医院耳鼻咽喉科,广东广州510515 [2]江门市中心医院耳鼻咽喉科,广东江门529000 [3]南方医科大学第三附属医院耳鼻咽喉科,广东广州510515

出　　处：《中华肿瘤防治杂志》2019年第19期1416-1424,共9页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金（81472534）;广东省自然科学基金（2015A030313237）

摘　　要：目的复发性难治性鼻咽癌治疗难度大,肿瘤干细胞是肿瘤复发和转移的根源。CD47是新近发现的重要免疫检查点,CD47抗体被认为是极具前景的抗肿瘤新药。本研究探讨CD47在鼻咽癌干细胞的表达及功能,为靶向CD47治疗复发性难治性鼻咽癌提供依据。方法无血清悬浮培养法处理鼻咽癌CNE2细胞,富集鼻咽癌干细胞。流式细胞术分选出CNE2/CD47+和CNE2/CD47-两群细胞,采用无血清悬浮成球培养、EdU增殖实验及平板克隆形成实验检测两群细胞在自我更新、体外增殖克隆能力的差异;实时荧光定量PCR及蛋白质印迹法分别检测两群细胞肿瘤干细胞标志物的mRNA、蛋白水平的表达。Balb/c裸鼠皮下成瘤实验评估两群细胞体内致瘤性的差异。结果 CNE2/CD47+组较CNE2/CD47-组形成肿瘤干细胞球的效率更高;CNE2/CD47+组的克隆形成能力强于CNE2/CD47-组,t=11.270,P<0.001;EDU增殖实验显示,CNE2/CD47+组的增殖率为(63.010±2.151)%,高于CNE2/CD47-组(36.390±1.925)%,差异有统计学意义,t=9.219,P<0.001。实时荧光定量PCR及蛋白质印迹结果显示,CNE2/CD47+组的肿瘤干细胞标志物表达更高,P<0.05。裸鼠皮下成瘤实验结果显示,CNE2/CD47+组肿瘤平均体积约(1 504.500±714.420)mm3,CNE2/CD47-组肿瘤平均体积约(471.800±502.700)mm3,差异有统计学意义,t=2.319,P<0.05。结论 CD47过表达可能是鼻咽癌干细胞形成和维持的关键因素,靶向CD47有可能成为治疗复发性难治性鼻咽癌的新途径。OBJECTIVE Relapsed or refractory nasopharyngeal carcinoma is difficult to treat.Cancer stem cells is considered as the root of recurrence and metastasis of nasopharyngeal carcinoma.CD47 is an important immune checkpoint recently discovered,and CD47 antibody is also considered as a promising new anti-tumor drug.The aim of the study was to investigate the expression and function of CD47 in nasopharyngeal carcinoma stem cells,and to provide evidence for targeting CD47 therapy in the treatment of recurrent and refractory nasopharyngeal carcinoma.METHODS The NPC stem cells were enriched from CNE2 by serum-free medium.The difference of self-renewal,proliferation and cloning ability between CNE2/CD47+ and CNE2/CD47-cell were tested by serum-free suspension sphere assay,EdU proliferation assay and plate cloning formation assay.Cancer stem cell markers were observed by qRT-PCR and Western blot assay.The xenograft tumor formation in Balb/C nude mice was used to evaluate the difference of tumorigenicity between the two groups of cells.RESULTS Compared with the CNE2/CD47-group,more tumorspheres formed in CNE2/CD47+ group.CD47 increased cloning formation(t=11.270,P<0.001)and proliferation ability of CNE2 cell(63.010±2.151)% vs(36.390±1.925)%,t=9.219,P<0.001 respectively,along with the raise of cancer stem cell markers(P<0.05).In vivo study also indicated that the volume of tumor in CNE2/CD47-positive group was significantly higher than CNE2/CD47-negative group[(1 504.5±714.42)mm3 vs(471.8±502.7)mm3,t=2.319,P<0.05].CONCLUSIONS CD47 overexpression is a critical factor of NPC stem cells formation and maintainess.Targeting CD47 may become a brand-new stratege of refractory nasopharyngeal carcinoma therapy.

关 键 词：CD47 鼻咽癌 肿瘤干细胞 自我更新 复发 

分 类 号：R739.6[医药卫生—肿瘤]