免疫毒素DTATEGF对人非小细胞肺癌脑转移瘤的影响及机制

Efficacy of bispecific targeted immunotoxin DTATEGF against NSCLC brain metastatic tumor PC9-BrM3 cells

目的:观察免疫毒素DTATEGF对体外培养的人NSCLC脑转移瘤细胞增殖、凋亡及其对肿瘤血管生成的影响。方法:MTT法检测不同浓度靶向毒素DTATEGF对体外培养的人NSCLC脑转移瘤PC9-BrM3细胞增殖的影响,流式细胞仪分析DTATEGF作用于PC9-BrM3细胞系48 h后细胞凋亡和细胞周期变化。12只皮下种植肿瘤的祼小鼠分为2组:瘤床内分别注入DTATEGF或对照液2μg,隔天一次,共5次,测量肿瘤体积及其微血管密度(MVD)。结果:DTATEGF明显抑制PC9-BrM3细胞的体外增殖,呈剂量依赖关系,其诱导PC9-BrM3细胞凋亡,1 pmol/L的DTATEGF作用PC9-BrM3细胞48 h后细胞凋亡率为(64.0±0.5)%,对照组为(1.5±0.4)%,差异有统计学意义(P<0.01);细胞周期检测显示:DTATEGF处理组SubG0/G1期和S期细胞别为(32.0±1.5)%和(2.0±0.4)%,而空白对照组分别为(5.0±0.6)%和(11.4±0.8)%,差异均有统计学意义(P<0.01)。动物实验显示DTATEGF处理组肿瘤体积较对照组生长缓慢,且DTATEGF处理组MVD为(15.6±4.6)/mm2,而空白对照组为(31.2±5.4)/mm2,差异均有统计学意义(P<0.05)。结论:DTATEGF抑制PC9-BrM3增殖、诱导细胞凋亡,明显抑制祼小鼠皮下种植的人NSCLC脑转移瘤细胞的生长及其新生血管的形成。

Objective： To investigate the in vitro and in vivo anticancer efficacy of the immunotoxin DTATEGF against human NSCLC brain metastatic tumor PC9-BrM3 cell line. Methods： The effect of the immunotoxin DTATEGF was tested for its ability to inhibit the proliferation of PC9-BrM3 cells in vitro by MTT assay. The cell cycle and the apoptosis of cells with 1 pmol/L DTATEGF were examined by flow cytometry. In vivo, 2 ug of DTATEGF or control Bickel3 was given intratumor to nude mice with established PC9-BrM3 xenografts on their hips, and tumor volumes were measured and tumor samples were investigated by immunchistochemistry SABC method, The microvessel density （MVD） was measured in each group. Results： In vitro, DTATEGF killed PC9-BrM3 cells and showed an IC50 of i pmol/L. The apoptotic rate in the 1 pmol/L DTATEGF group was （64.0±0.5）%, significantly higher than that in the control group （1.5±0.4）% （P〈0.01）. The cell cycle was obviously inhibited by DTATEGF in a dose-dependent manner. The percentage of cells treated with 1 pmol/L DTATEGF in SubG0/G1 phase was （32.0± 1.5）%, significantly higher than that in the control group （2.0±0.4） % （P〈0.01）. In vivo, DTATEGF significantly inhibited the growth of PC9-BrM3 hip tumors （P〈0.05）. The MVD of the DTATEGF group was （15.6±4.6）/mm2, significantly lower than that of the control group （31.2±5.4）/mm2 （P〈 0.01 ）. Conclusion： DTATEGF inhibits the growth of the PC9-BrM3 cell line and induces its apoptosis. It is highly efficacious against human metastatic NSCLC brain tumor and against neovascularization.