白细胞介素2基因转染细胞因子诱导的杀伤细胞对恶性黑素瘤细胞的杀伤作用  

Cytotoxic effects of cytokine- induced killer cells transfected with the interleukin- 2 gene on malignant melanoma cells

作　　者：芦兰[1] 谢丛华[1] 张浩中 许绿叶 师幸伟 谢君 车彪 丁雯 

机构地区：[1]武汉大学中南医院肿瘤放化疗科,430071 [2]长江航运总医院内科 [3]长江航运总医院外科

出　　处：《中华皮肤科杂志》2017年第4期257-262,共6页Chinese Journal of Dermatology

基　　金：武汉市卫计委临床医学科研课题（WX13D16）

摘　　要：目的 探讨白细胞介素2（IL-2）基因转染细胞因子诱导的杀伤细胞（CIK）对恶性黑素瘤的杀伤能力。方法 提取小鼠脾细胞，分离淋巴细胞，培养CIK细胞，用携带IL-2的质粒PEGF-N1-IL-2转染CIK细胞，荧光显微镜观察质粒转染情况，反转录-聚合酶链反应（RT-PCR）鉴定IL-2基因的表达。将效应细胞（CIK细胞或IL-2转染CIK细胞）和靶细胞（B16黑素瘤细胞）分别按照效靶比10∶1、20∶1和40∶1混合培养，采用4 h乳酸脱氢酶释放测定法，检测两种CIK细胞对B16细胞的细胞毒活性。按照效靶比40∶1混合，采用酶联免疫吸附测定法（ELISA）检测两种CIK细胞IL-2、干扰素γ（IFN-γ）和肿瘤坏死因子α（TNF-α）水平。建立小鼠黑素瘤模型，将28只模型小鼠平均分为4组：对照组（瘤旁注射0.2 ml生理氯化钠溶液）、IL-2组（瘤旁注射100 IU IL-2）、CIK组（瘤旁注射细胞数约1 × 106 CIK细胞悬液）、IL-2转染CIK组（瘤旁注射细胞数约1 × 106 IL-2转染CIK细胞悬液），通过肿瘤形态学和抑瘤率、细胞凋亡率来评价荷瘤小鼠肿瘤生长情况。两组正态分布计量资料的比较行t检验，多组计量资料的比较采用方差分析，两两间多重比较采用LSD-t检验。结果 荧光显微镜及RT-PCR均显示IL-2转染CIK细胞成功。效靶比实验显示，40∶1时IL-2转染CIK细胞对B16细胞的毒性最强，IL-2转染CIK细胞组分泌IL-2（1107.26 ± 6.49 pg/ml）、IFN-γ（50.01 ± 3.35 pg/ml）和TNF-α（39.86 ± 3.25 pg/ml）的能力明显高于CIK细胞组（分别为51.09 ± 3.85、32.71 ± 2.43、30.11 ± 3.08 pg/ml），两组比较，t值分别为442.60、14.93和6.89，差异均有统计学意义（P 〈 0.01）。动物实验显示，与干预前相比，干预后对照组小鼠肿瘤体积明显增大（P 〈 0.05），而IL-2组、CIK组和IL-2转染CIK组小鼠肿瘤体积明显减小（P 〈 0.001），且IL-2转染CIK组肿瘤体积显著小于其他3组�Objective To evaluate cytotoxic effects of cytokine-induced killer cells （CIK cells） transfected with the interleukin-2 （IL-2） gene on malignant melanoma cells. Methods Mouse spleen cells were extracted, lymphocyte cells were separated, and CIK cells were prepared from these lymphocyte cells. PEGF-N1 plasmids containing IL-2 gene （PEGF-N1-IL-2） were transfected into CIK cells. Fluorescence microscopy was used to observe transfection products, and reverse transcriptase-polymerase chain reaction （RT-PCR） was conducted to determine the IL-2 mRNA . Then, effector cells such as CIK cells and IL-2-transfected CIK cells were separately co-cultured with target cells （B16 melanoma cells） at effector-target ratios of 10∶1, 20∶1 and 40∶1, then 4-hour lactate dehydrogenase release assay was performed to evaluate cytotoxic effects of the two kinds of CIK cells on B16 cells. After effector cells were co-cultured with target cells at the effector-target ratio of 40∶1 for 48 hours, enzyme-linked immunosorbent assay （ELISA） was conducted to detect levels of IL-2, interferon-γ （IFN-γ） and tumor necrosis factor-α （TNF-α） in the supernatant of the two kinds of CIK cells. Finally, mouse models of melanoma were established, and a total of 28 melanoma-bearing mice were divided into 4 groups to be peritumorally injected with 0.2 ml sodium chloride physiological solution （control group）, 100 IU IL-2 solution （IL-2 group）, CIK cell suspension at a cell density of 1 × 106 cells per milliliter （CIK group） and IL-2-transfected CIK cell suspension at a cell density of 1 × 106 cells per milliliter （IL-2-transfected CIK group） respectively. Tumor morphology, tumor inhibition rate and cell apoptosis rate were used to evaluate tumor growth in the above groups. If data were normally distributed, t-test was used for comparing means between two groups, and analysis of variance and least significant difference （LSD）-t test were used for comparing means among multiple groups. Results Fl

关 键 词：白细胞介素2 细胞因子诱导杀伤细胞 转染 黑色素瘤 

分 类 号：R739.5[医药卫生—肿瘤]