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目的:探讨细胞因子诱导的杀伤细胞(cytokine-induced killer cells,CIKs)对宫颈癌CasKi和Hela细胞的体内外杀伤效应。方法:从宫颈癌患者的外周血分离单个核细胞,以改良方法(不加IFN-γ,只加IL-2和anti-CD3抗体)刺激扩增得到CIKs,用流式细胞仪分析CIKs表型。ELISA法检测CIKs培养上清中IFN-γ、IL-2和TNF-α的分泌水平。LDH释放法检测CIKs对CasKi和HeLa细胞的杀伤作用。建立荷CasKi或Hela细胞小鼠模型,以1×106或1×107个CIKs小鼠静脉注射治疗,每周1次,连续治疗3周,检测小鼠移植瘤的体积和重量。结果:在抗CD3抗体和IL-2的刺激下,宫颈癌患者外周血单个核细胞被诱导培养成CIKs,其中CD3+CD56+细胞得到大量扩增。PHA激活后,CIKs产生大量的IFN-γ和TNF-α,而IL-2的分泌量较少。被激活的CIKs可显著地杀伤宫颈癌CasKi细胞和HeLa细胞,最大杀伤率分别达(43.2±1.8)%和(46.2±1.5)%,且呈剂量依赖性。体内抗肿瘤实验结果显示,CIKs能显著抑制小鼠皮下宫颈癌移植瘤的生长(P<0.01)。结论:本实验的改良方法能有效制备CIKs,该CIKs在体内外对宫颈癌CasKi和Hela细胞有明显的杀伤效应。
Objective: To investigate the cytotoxicity of cytokine-induced killer cells (CIKs) on cervical cancer cells CasKi and Hela cells in vitro and in vivo. Methods: Mononuclear cells were isolated from the peripheral blood of patients with cervical cancer and CIKs were obtained by ameliorative methods (without IFN-γ plus IL-2 and anti-CD3 antibodies only). The CIKs phenotypes were analyzed by flow cytometry . The secretion of IFN-γ, IL-2 and TNF-α in the culture supernatants of CIKs were detected by ELISA. LDH release assay CIKs on the killing of CasKi and HeLa cells. The mouse model of CasKi or Hela cells was established. The mice were treated with 1 × 10 6 or 1 × 10 7 CIKs intravenously once a week for 3 weeks. The volume and weight of the transplanted tumor were measured. RESULTS: Peripheral blood mononuclear cells were induced to grow into CIKs under the stimulation of anti-CD3 antibody and IL-2, and CD3 + CD56 + cells were greatly expanded. After PHA activation, CIKs produced large amounts of IFN-γ and TNF-α, while IL-2 secreted less. Activated CIKs could significantly kill cervical cancer cells and HeLa cells, with the maximum killing rates of (43.2 ± 1.8)% and (46.2 ± 1.5)%, respectively, in a dose-dependent manner. In vivo anti-tumor experiment results showed that CIKs could significantly inhibit the growth of transplanted subcutaneous cervical cancer in mice (P <0.01). Conclusion: The improved method of this experiment can effectively prepare CIKs, the CIKs in vitro and in vivo of cervical cancer CasKi and Hela cells have obvious killing effect.