IL-2联合IL-15提高肾母细胞瘤患儿外周血NK细胞对肿瘤细胞的杀伤作用  

Interleukin-2 and Interleukin-15 enhance the cytotoxicity of NK cell against tumor in peripheral blood of Wilms tumor children

作　　者：石秦林 林涛[1] 沈炼桔[1] 刘丰[1] 张德迎[1] 陆鹏[1] 刘星[1] 何大维[1] 李旭良[1] 魏光辉[1] Shi Qinlin;Lin Tao;Sheng Lianju;Liu Feng;Zhang Deying;Lu Peng;Liu Xing;He Dawei;Li Xuliang;We Guanghui(Pediatric Research Institute, Children＇s Hospital, Chongqing Medical University /Chongqing Key Laboratory of Pediatrics/Ministry of Education Key Laboratory of Child Development Disorders~China International Science ＆ Technology Cooperation Base of Child Development ＆ Critical Disorder;Department of Urology, Affiliated Children＇s Hospital, Chongqing Medical University, Chongqing 400014, China)

机构地区：[1]重庆医科大学附属儿童医院泌尿外科、儿童发育疾病研究教育部重点实验室、重庆医科大学附属儿童医院儿科研究所、儿童发育重大疾病国家国际科技合作基地、儿科学重庆市重点实验室、儿童泌尿生殖发育与组织工程重点实验室,400014

出　　处：《中华小儿外科杂志》2018年第5期345-350,共6页Chinese Journal of Pediatric Surgery

基　　金：国家级临床重点专科项目[2013（544）]

摘　　要：目的 观察肾母细胞瘤（WT）患儿外周血NK细胞经IL-2、ILd5扩增后对肾母细胞瘤SK-NEP-1的杀伤作用.方法 以8例WT患儿作为病例组,8例健康体检儿童作为对照组.采集每例外周血4 ml,按Fieoll法分离外周血单个核细胞（PBMC）,流式细胞仪检测、分选两组PBMC中CD3＋ CD16＋ CD56＋ NK细胞;另采集病例组和对照组外周血4 ml,分离PBMC后,分两组进行培养[IL-2（100 U/ml）培养组、IL-2（100 U/ml）联合IL-15（10 ng/ml）培养组],流式细胞仪检测、分选培养后CD3-CD16＋ CD56＋ NK细胞.CCK8法检测在不同效靶比（E∶T分别为5∶1、10∶1、20∶1）（效应细胞：NK细胞;靶细胞：SK-NEP-1）情况下,WT患儿外周血NK细胞经IL-2、IL-15扩增后对SK-NEP-1细胞的杀伤作用.western blot检测培养前、IL-2培养组、IL-2联合IL-15组CD3-、CD16＋、CD56＋、NK细胞中TNF-α及TNF-β表达情况.结果 病例组和对照组采用IL-2联合IL-15扩增后的NK细胞比例分别增加[（7.3±2.4）％比（21.2±3.2）％],[（4.5±1.6）％比（10.1±3.4）％];数量分别增多（70.60±22.5）,（30.12±33.16）倍,（P＜0.05）.E∶T为10∶1、20∶1时,与单用IL-2扩增比较,IL-2联合IL-15扩增组的NK细胞对肾母细胞瘤细胞的杀伤作用均增强[（16.3±2.1）比（10.5±1.6）]、[（23.9±3.2）比（13.7±2.5）],（P＜0.05）.病例组患儿外周血CD3 CD16＋ CD56＋ NK细胞采用IL-2联合IL-15培养后TNF-α和TNF-β的表达量,较IL-2组、培养前,均显著增加（P＜0.05）.结论 IL-2联合IL-15能够提高患儿外周血NK细胞的比例及数量,增加其分泌TNF的能力,从而提高其对肿瘤的杀伤作用.Objective To observe the effect of IL-2 and IL-15 culturing with （NK） natural killer cell of peripheral blood in vitro and improve the cytotoxicity of NK cell against Wilms tumor cell line SK-NEP-1.Methods Eight patients with Wilms tumor （experiment group） and 8 normal children （control group） were recruited.For each case,peripheral blood （4 ml） was collected and mononuclear cell （PBMC） separated with Ficoll reagent.CD3-CD16＋ CD56＋ NK cells were detected and sorted by flow cytometry.In addition,another acquisition case and control groups in peripheral blood of 4 ml cultured with IL-2（100 U/ml） or IL-2 （100 U/ml） plus IL-15 （10 ng/ml）,sorting after culturing of CD3-CD16＋ CD56＋ NK cells.CCK8 method was used for detecting the effect of different effector cells and target cell ratio （E∶ T 5 ∶ 1,10∶1,20 ∶ 1） （effector cells,NK cells ＆ target cells：SK-NEP-1） cases,the killing effect of WT with peripheral blood NK cells （cultured with IL-2,IL-15） to SK-NEP-1 cells.The expressions of TNF-α and TNF-γ of CD3 CD16＋ CD56＋ NK cells （pre-culture,IL-2 group,IL-2 ＆ IL-15 groups） were detected by western blot.Results The proportion of NK cell in two group,using IL-2 and IL-15 significantly increased than before [case group：（7.3 ± 2.4）％ vs （21.2 ± 3.2） ％],control group [（4.5 ± 1.6） ％ vs （1 0.1 ± 3.4） ％] and quantity increased respectively （70.60 ± 22.5）,（30.12± 33.16） times （P＜0.05）.After using IL-2 and IL-15,cytotoxicity of NK cell against Wilms tumor cell line （10∶1 20∶1） obviously increased [（16.3 ± 2.1） vs （10.5 ± 1.6）],[（23.9 ± 3.2） vs （13.7 ± 2.5）] （P＜0.05）.The expressions of TNF-α and TNF-γ （post-culture） increased than （pre-culture,IL-2 group）and significantly in IL-2 plus IL-15 group （P＜0.05）.Conclusions The quantity and proportion of NK cell were improved among PBMCs by IL-2 plus IL-15.Also there is enhanced cytotoxicity of NK cell against Wilms tumor

关 键 词：肾母细胞瘤 自然杀伤性细胞 白细胞介素2 白介素15 儿童 

分 类 号：R737.11[医药卫生—肿瘤]