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目的:探讨细胞因子诱导的杀伤细胞(cytokine-induced killer cell,CIK)对荧光素酶标记的人宫颈癌HeLa-luc细胞的体内外抗肿瘤作用,了解CIK回输荷瘤小鼠后在不同器官的分布特点。方法:由健康志愿者外周血单个核细胞体外诱导培养CIK,流式细胞术检测CIK表面分子的表达,RT-PCR法检测IFN-γmRNA的表达,MTT法和瑞氏-姬姆萨染色测定CIK对HeLa-luc细胞的杀伤作用。建立荷HeLa-luc瘤BALB/c裸鼠模型,体内成像系统观察荷瘤小鼠肿瘤大小变化及CIK治疗效果,共聚焦显微镜观察不同器官中CIK的分布情况。结果:CIK在体外诱导培养14～21 d,其生长达到高峰,此时CD3+CD56+T细胞的比例增加50倍以上,IFN-γmRNA表达水平也达到高峰。在效靶比为20∶1、40∶1时,CIK对HeLa-luc细胞的杀伤率分别为(51.16±2.64)%、(72.14±4.21)%,明显高于对照组的(16.33±3.09)%、(21.26±2.71)%(P<0.05)。CIK治疗5周和8周后,对荷瘤小鼠的抑瘤率分别为47.18%、64.38%。CIK治疗组荷瘤小鼠外周血中IFN-γ水平为(61.92±6.49)pg/ml,明显高于对照组的(34.30±1.78)pg/ml(P<0.05)。CFSE标记的CIK经腹腔和瘤旁注射入荷瘤小鼠3 h,在肺、肝、脾、外周血、肿瘤中均可观察到绿色荧光;腹腔途径注射24 h时,肿瘤组织中CIK浓度达到高峰(20.56±1.72)%;瘤旁途径注射3 h时,肿瘤组织中CIK达到高峰(25.75±3.45)%。结论:CIK在体内外对宫颈癌HeLa-luc细胞均有较强的杀伤作用,CIK经不同途径注射荷瘤小鼠后可以广泛分布于全身器官,其到达各脏器的浓度与输注途径及时间密切相关。
Objective: To investigate the anti-tumor effects of cytokine-induced killer cells (CIK) on luciferase-labeled human cervical cancer HeLa-luc cells in vitro and in vivo. Distribution characteristics. Methods: Peripheral blood mononuclear cells from healthy volunteers were used to induce CIK. Flow cytometry was used to detect the expression of CIK surface molecules. The expression of IFN-γ mRNA was detected by RT-PCR. MTT and Wright-Giemsa staining were used to determine CIK HeLa-luc cell killing effect. BALB / c nude mice model was established by HeLa-luc tumor. The in vivo imaging system was used to observe tumor size change and CIK treatment effect in tumor-bearing mice. Confocal microscopy was used to observe the distribution of CIK in different organs. Results: The CIK cells were cultured in vitro for 14-21 days and their growth peaked. At this time, the proportion of CD3 + CD56 + T cells increased more than 50 folds, and the expression level of IFN-γ mRNA also peaked. The killing rates of CIK on HeLa-luc cells were (51.16 ± 2.64)% and (72.14 ± 4.21)%, respectively, which were significantly higher than those of the control group (16.33 ± 3.09) %, (21.26 ± 2.71)% (P <0.05). After 5 and 8 weeks of CIK treatment, the tumor-inhibiting rates of tumor-bearing mice were 47.18% and 64.38%, respectively. The level of IFN-γin peripheral blood of mice bearing CIK was (61.92 ± 6.49) pg / ml, which was significantly higher than that of the control group (34.30 ± 1.78) pg / ml (P <0.05). CFSE-labeled CIK was injected into the tumor-bearing mice 3 h after intraperitoneal and para-tumorous injection, and green fluorescence was observed in lung, liver, spleen, peripheral blood and tumor. At 24 h after intraperitoneal injection, the CIK concentration in tumor tissue peaked (20.56 ± 1.72)%. CIH reached the peak (25.75 ± 3.45)% in tumor tissue at 3 h after intratumoral injection. Conclusion: CIK can kill cervical cancer HeLa-luc cells both in vitro and in vivo. CIK can be widely distributed in whole body organs after being injected into tumor-bearing mice via various routes. Time is closely related.