高效扩增和活化nk细胞并对骨髓瘤细胞有特异性杀伤作用.docVIP

编辑部老师： 您好！ 我已经按审稿意见提供了伦理委员会批准件， 并在修改稿中增补了5例健康志愿者和乳腺癌患者的信息。 谢谢！ 施菊妹 NK细胞对人乳腺癌细胞株杀伤作用及机制研究 祖卫兵 基金项目：国家自然科学基金作者单位：226100 江苏省海门市人民医院普外科祖卫兵 通信作者：电子邮箱为shijumei@ 【摘要 目的 观察体外扩增自然杀伤（ＮＫ）细胞的表型，并研究其对不同乳腺癌细胞株的杀伤作用及机制。方法 抽取志愿者外周血５０ ｍＬ，分离单个核细胞，与基因修饰K562细胞共培养，采用流式细胞仪分析和Calcein释放法，检测NK细胞的表型，分析NK细胞对MDA-MB231和MCF-7乳腺癌细胞的杀伤率及作用机制。 结果 扩增后NK细胞活化分子NKG2D、NKp30、NKp44和DNAM-1表达增强；扩增后NK细胞对乳腺癌细胞的杀伤率分别为61.7%（MDA-MB231）和66.1%（MCF-7）（效/靶比为20:1），对照组扩增前NK细胞的杀伤率分别为28.2%（MDA-MB231）和29.6%（MCF-7）；扩增后NK细胞杀伤率增强与高表达NKG2D、NKp30、NKp44和DNAM-1活化受体相关。结论 扩增后NK细胞表达部分活化分子增强，其对乳腺癌细胞杀伤作用明显增强，扩增后NK细胞具有增强的抗肿瘤活性可能和这些活化分子高表达有关。 关键词 自然杀伤细胞; 扩增; 体外；乳腺癌 Study of the role and mechanism of natural killer cells with specific cytotoxicity against breast cancer cells in vitro ZU Weibing, GAO Minjie, JIANG Weixing, ZHU Feng，SHI Jumei. *Department of General Surgery, Haimen People’s Hospital, Jiangsu, 226100, China Corresponding author: SHI Jumei . E-mail: shijumei@ 【Abstract】 Objective This study is to investigate the phenotype of expanded human NK cells in vitro. We also tested the role of expanded NK cells in killing of different breast cancer cells and its mechanism. Methods Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood donored by 5 healthy individuals. PBMC were co-cultured with irradiated genetic modified K562 cell transfectants. The cultures were then analyzed for phenotype and cytotoxicity by flow cytometry and calcein-release assays. Results We observed stronger expression of activating receptor NKG2D,NKp30,NKp44,DNAM-1 in expanded NK cells than in non-expanded NK cells. The expanded NK cells killed MDA-MB231 and MCF-7 avidly as shown by calcein assays. The mean cytotoxic rate of expanded NK cells against breast cancer cells was 61.7% (MDA-MB231), 66.1% (MCF-7) at an E:T ratio of 20:1 compared to 28.2% (MDA-MB231), 29.6% (MCF-7) of non-expanded NK cells, respectively. The enhanced killing of breast cancer cells by expanded NK cells was related to the higher expression o