NK细胞对人肝癌细胞的杀伤作用及其可能机制  

Specific cytotoxicity of natural killer cells against hepatocellular carcinoma and potential mechanism

作　　者：王文秀 习佳飞[2,3] 赵月 何丽娟 周军年[2,3] 范增 张彪[2,3] 王海洋 曾泉[2,3] 南雪 岳文[1,2,3] 裴雪涛 

机构地区：[1]广西医科大学,南宁530021 [2]军事医学科学院野战输血研究所,全军干细胞与再生医学重点实验室,血液安全保障技术研究北京市重点实验室,北京100850 [3]华南干细胞与再生医学研究中心,广州510005

出　　处：《军事医学》2017年第9期775-783,共9页Military Medical Sciences

基　　金：国家重点研发计划资助项目(2017YFA0103100,2017YFA0103103,2017YFA0103104);广州市健康医疗协同创新重大专项资助项目(201704020224);国家自然科学基金资助项目(81773137)

摘　　要：目的以天然杀伤细胞(NK)-92细胞系为模型,研究NK细胞对肝癌细胞的杀伤作用,并探讨其可能的作用机制。方法建立NK-92细胞系的体外培养方法,利用酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)和乳酸脱氢酶细胞毒性检测实验(lactate dehydrogenase cytotoxicity assay)等体外实验验证NK-92细胞对多种肝癌细胞的体外杀伤作用。同时建立人肝癌细胞株HepG2的裸鼠皮下荷瘤模型,每只裸鼠分别在荷瘤后第2、9、16、23天共4次通过尾静脉注射1×10~7个NK-92细胞,对照组注射等体积PBS,密切观察实验组和对照组裸鼠移植瘤大小、体积、质量以及裸鼠存活状态。荷瘤30 d后活杀裸鼠,取肿瘤组织进行大体观察,并对肿瘤组织切片进行HE染色和免疫荧光检测,明确瘤内磷脂酰肌醇蛋白聚糖-3(glypican-3,GPC3)蛋白的表达。结果体外长期培养的NK-92细胞能保持典型的NK细胞表型特征,细胞活性好,利用ELISA和LDH实验对NK-92细胞的体外杀瘤能力进行鉴定。结果表明,其对多种肝癌细胞系均具有很好的杀伤作用。裸鼠抑瘤实验结果显示,体内NK-92细胞对肝癌组织有较好的杀伤作用,实验组肝癌组织体积明显小于对照组,且肝癌组织免疫组化和免疫荧光结果均表明GPC3表达明显降低。初步显示,NK细胞可能通过杀伤GPC3阳性肝癌细胞来发挥其治疗作用。结论NK细胞治疗有望作为有效的肝癌治疗手段。该研究利用NK-92细胞系为研究对象,证明其在体外和体内均对肝癌细胞有很好的杀伤作用,同时初步表明其对肝癌的杀伤作用机制可能是通过杀伤肝癌组织内的GPC3阳性细胞;该研究对进一步提高NK细胞对肝癌的治疗作用及明确其作用机制奠定了基础。NK细胞除抗肿瘤作用外对多种细菌和病毒等病原体有很好的杀伤作用,同时对进一步拓展NK细胞的应用范围提供了参考。Objective To evaluate the cytotoxicity of natural killer（NK）-92 cell lines against various human hepatocellular carcinoma cells（HCCs） and to explore the potential mechanism.Methods We established a culture method of NK-92 cell lines in vitro.Lactate debydrogenase（LDH） cytotoxicity assays and cytokine release assays were performed to determine whether NK-92 cell lines could recognize and kill HCCs in vitro.At the same time,Nu/Nu mices were housed.Subcutaneous（sc） xenografts HepG2 models of human hepatocellular carcinoma were established.1 × 107 NK-92 cells were intravenously（iv） injected through the tail vein on days 2,9,16,23 while the control group was injected with PBS in the same way.Tumor size,tumor volume,tumor mass and mouse survival status were closely observed in experimental and control groups.Mice were euthanized when tumor-bearing time reached 28 days.Xenograft tissues were taken for general observation.Sections were cut and processed for HE staining and immunofluorescence staining.The expression of glypican-3（GPC3） protein in xenografts tissue was clearly defined.Results NK-92 cell lines that were chronically cultured in vitro and maintained typical phenotypic characteristics of NK cells with good cellular activity.Enhanced cytotoxicity and IFN-γproduction of NK-92 cell lines were identified by LDH and ELISA,indicating that NK-92 cell lines could recognize and kill different kinds of HCCs.In addition,NK-92 cell lines efficiently suppressed the growth of HCC xenografts in vivo.Tumor volume in experimental group was significantly reduced compared with control group and there was low a GPC3 expression in experimental group through immunofluorescence and immunohistochemistry results,pointing to the possibility that the cytotoxicity of NK cells was correlated with GPC3~＋ HCCs.Conclusion NK cells provide a promising means of therapeutic intervention for HCCs.NK-92 cell lines could eliminate HCC cells in vitro and in vivo.The cytotoxicity of NK-92 cell lines may work by killing th

关 键 词：杀伤细胞 天然 癌细胞 肝 细胞毒作用 磷脂酰肌醇蛋白聚糖类 

分 类 号：R735.7[医药卫生—肿瘤]