白细胞介素-12对肿瘤切除术后肿瘤复发抑制作用的实验研究

EXPERIMENTAL STUDY ON INHIBITORY EFFECT OF INTERLEUKIN-12 ON TUMOR RECURRENCE AFTER TUMOR RESECTION

目的探讨白细胞介素-12(IL-12)对肿瘤切除术后肿瘤复发的抑制作用及其作用机制。方法建立BALB/C小鼠和裸鼠术后残余肿瘤模型,将其随机分为模型对照组(生理盐水0.1 mL)、阳性对照组(长春新碱10 mg/kg)、IL-12低剂量组(重组鼠源IL-1225 ng/kg)、IL-12中剂量组(重组鼠源IL-1250 ng/kg)、IL-12高剂量组(重组鼠源IL-12100 ng/kg),每组11只,每2 d给药1次,共治疗15 d。BALB/C小鼠每2 d称体质量1次,于末次给药24 h后腹主动脉取血。BALB/C小鼠和裸鼠处死后分别剥离残余肿瘤组织并称质量;采用ELISA法检测BALB/C小鼠血清及肿瘤组织中干扰素-γ(IFN-γ)的浓度,HE染色分析肿瘤及各主要脏器组织的病理特征。以不同浓度重组鼠源IL-12(rmIL-12)处理鼠源性腹腔巨噬细胞Ana-1、人源性肺癌细胞A549、人脐静脉内皮细胞HUVEC,采用MTT法检测细胞的增殖能力,采用ELISA法检测巨噬细胞Ana-1上清液中肿瘤坏死因子-α(TNF-α)浓度。结果给药15 d后,析因设计方差分析结果显示,小鼠种类对残余肿瘤质量有影响(F_(小鼠种类)=52.75,P<0.05),分组对残余肿瘤质量有影响(F_(分组)=31.08,P<0.05),小鼠种类和分组无交互作用(P>0.05),单独效应结果显示,与模型对照组比较,IL-12各给药组中BALB/C小鼠、裸鼠的肿瘤质量均显著降低(F=20.35、12.94,P<0.05),除模型对照组外,BALB/C小鼠残余肿瘤质量均低于裸鼠(F=7.92~24.07,P<0.05)。HE染色结果显示,IL-12各给药组BALB/C小鼠主要脏器与模型对照组比较未见明显病理变化,但肿瘤组织出现大量空泡,炎性浸润区域明显增多。IL-12各给药组BALB/C小鼠血清IFN-γ浓度与模型对照组比较差异无显著性(P>0.05);IL-12各给药组BALB/C小鼠肿瘤组织中IFN-γ的浓度与模型对照组比较显著升高(F=31.32,P<0.05)。体外实验结果显示,随着rmIL-12浓度升高,巨噬细胞Ana-1增殖能力升高(q=8.63~24.42,P<0.05),分泌TNF-α浓度增加(q=4.49~18.93,P<0.05)。结论IL-12可能是通过调节肿瘤微环境抑制肿瘤切除后残余肿瘤生长,但对机体无明显不良反应,为IL-12临床药物的开发提供了数据参考。

Objective To investigate the inhibitory effect of interleukin-12(IL-12)on tumor recurrence after tumor resection and its mechanism.Methods BALB/C mice and nude mice were used to establish a model of postoperative residual tumor,and then they were randomly divided into model control group(normal saline 0.1 mL),positive control group(vincristine 10 mg/kg),low-dose IL-12 group(recombinant murine IL-1225 ng/kg),middle-dose IL-12 group(recombinant murine IL-1250 ng/kg),and high-dose IL-12 group(recombinant murine IL-12100 ng/kg),with 11 animals in each group.The drugs were administered once every 2 d for 15 d in total.BALB/C mice were weighed once every 2 d,and blood samples were collected from the abdominal aorta at 24 h after the last administration.Residual tumor tissue was stripped and weighed after BALB/C mice and nude mice were sacrificed;ELISA was used to measure the concentration of interferon-γ(IFN-γ)in the serum and tumor tissue of BALB/C mice;HE staining was used to observe the pathological features of tumor and main visceral organs.After mouse-derived abdominal cavity macrophage cell line Ana-1,human lung cancer cell line A549,and human umbilical vein endothelial cells(HUVECs)were treated with recombinant murine IL-12 at different concentrations,MTT assay was used to measure cell proliferative capacity,and ELISA was used to measure the concentration of tumor necrosis factor-α(TNF-α)in the supernatant of Ana-1 macrophages.Results After 15 d of administration,the factorial analysis of variance showed that mouse species had a certain inf-luence on the weight of residual tumor(F_(mouse species)=52.75,P<0.05)and grouping also had an influence on the weight of residual tumor(F_(grouping)=31.08,P<0.05),while there was no interaction between mouse species and grouping(P>0.05).The simple effect analysis showed that compared with the model control group,the IL-12 administration groups had a significant reduction in tumor weight in both BALB/C mice and nude mice(F=20.35,12.94,P<0.05),and for all groups except the model control group,the weight of residual tumor in BALB/C mice was significantly lo-wer than that in nude mice(F=7.92-24.07,P<0.05).HE staining showed that compared with the model control group,the IL-12 administration groups had no significant pathological changes of the main visceral organs of BALB/C mice,but with a large number of vacuoles in tumor tissue and a significant increase in inflammatory infiltration area.There was no significant difference in serum IFN-γconcentration in BALB/C mice between the IL-12 administration groups and the model control group(P>0.05),and compared with the model control group,the IL-12 administration groups had a significantly higher concentration of IFN-γin the tumor tissue of BALB/C mice(F=31.32,P<0.05).In vitro experiments showed that the proliferative capacity of Ana-1 macrophages(q=8.63-24.42,P<0.05)and the concentration of secreted TNF-α(q=4.49-18.93,P<0.05)increased with the increase in the concentration of recombinant murine IL-12.Conclusion IL-12 may inhibit residual tumor growth after tumor resection by regulating tumor microenvironment,with no significant adverse effects on the body,which provides a data reference for the development of IL-12 drugs.