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

摘要/Abstract

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（均P < 0.01），但IL?2组和CIK组差异无统计学意义（P > 0.05）。CIK组、IL?2组和IL?2转染CIK组的细胞凋亡率均显著大于对照组（P < 0.01），IL?2转染CIK组的细胞凋亡率及抑瘤率均显著大于IL?2组和CIK组（P < 0.01），而IL?2组和CIK组细胞凋亡率及抑瘤率差异无统计学意义（P > 0.05）。结论 IL?2转染CIK细胞对恶性黑素瘤有更强的杀伤作用。

Abstract: Lu Lan, Xie Conghua, Zhang Haozhong, Xu Lyuye, Shi Xingwei, Xie Jun, Che Biao, Ding Wen Department of Radiation and Chemotherapy, Zhongnan Hospital of Wuhan University, Wuhan 430071, China （Lu L, Xie CH）; Department of Internal Medicine, General Hospital of the Yangtze River Shipping, Wuhan 430010, China （Zhang HZ, Xu LY, Shi XW, Ding W）; Department of Surgery, General Hospital of the Yangtze River Shipping, Wuhan 430010, China （Xie J, Che B） Corresponding author: Xie Conghua, Email: chxie_65@whu.edu.cn 【Abstract】 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 Fluorescence microscopy and RT?PCR both showed that IL?2 was successfully transfected into CIK cells. The cytotoxic effect of IL?2?transfected CIK cells on B16 cells was strongest at the effector?target ratio of 40∶1. Levels of IL?2, IFN?γ and TNF?α were also significantly higher in the supernatant of IL?2?transfected CIK cells ［（1107.26 ± 6.49） pg/ml, （50.01 ± 3.35） pg/ml, （39.86 ± 3.25） pg/ml］ than those in that of CIK cells ［（51.09 ± 3.85） pg/ml, （32.71 ± 2.43） pg/ml, （30.11 ± 3.08） pg/ml, t = 442.60, 14.93, 6.89, all P < 0.01］. Animal experiments showed that the tumor volume obviously increased in the control group （P < 0.05）, but significantly decreased in the IL?2 group, CIK group and IL?2?transfected CIK group （all P < 0.001） after intervention compared with those before intervention. Furthermore, the tumor volume in the IL?2?transfected CIK group was significantly less than that in the other three groups （all P < 0.01）, but no significant difference was observed between the IL?2 group and CIK group （P > 0.05）. In addition, the apoptosis rate was significantly higher in the IL?2 group, CIK group, and IL?2?transfected CIK group than that in the control group （all P < 0.01）. The apoptosis rate and tumor inhibition rate were significantly higher in the IL?2?transfected CIK group than those in the IL?2 group and CIK group （all P < 0.01）, but insignificantly different between the IL?2 group and CIK group （P > 0.05）. Conclusion IL?2?transfected CIK cells had stronger killing effects on malignant melanoma.

芦兰谢从华张浩中许绿叶谢君车彪师幸伟. 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[J]. 中华皮肤科杂志, 2017, 50(4): 257-262.doi:

lu-lan 师幸伟. Cytotoxic effects of cytokine?induced killer cells transfected with the interleukin?2 gene on malignant melanoma cells[J]. Chinese Journal of Dermatology, 2017, 50(4): 257-262.doi:

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