三氧化二砷通过抑制新生血管形成发挥抗小鼠恶性黑色素瘤作用

The anti-tumor effects of arsenic trioxide on murine B16 malignant melanoma via inhibition of neovascularization

目的：采用小动物活体成像技术研究三氧化二砷（As2O3）的抗小鼠恶性黑色素瘤作用及其机制。方法：荧光素酶（luciferase,Luc）基因标记小鼠B16黑色素瘤（B16-Luc）细胞,MTT比色法和Annexin V/PI双标记法分别检测细胞增殖活性及凋亡;BALB/c小鼠腋下接种B16-Luc细胞制备荧光黑色素瘤动物模型,腹腔注射4和8 mg/kg As2O3治疗25天,小动物光学活体成像系统连续动态观察肿瘤生长;治疗末期处死动物、剥离肿瘤块、制片,HE和CD34免疫组化染色观察肿瘤病理和新生微血管形成。结果：1.0~32.0μmol/L As2O3体外显著抑制B16-Luc细胞增殖和诱导其凋亡。活体成像技术观察显示As2O3在体内剂量依赖性地抑制小鼠B16-Luc恶性黑色素瘤的生长,肿瘤组织中新生微小血管显著减少,肿瘤组织中心呈明显坏死改变。结论：As2O3体外诱导小鼠恶性黑色素瘤B16细胞凋亡,主要通过降低肿瘤组织新生血管的形成、促使肿瘤坏死而发挥抗恶性黑色素瘤作用。

Objective： To study the anti-proliferating action of arsenic trioxide（ As2O3） on murine B16 malignant melanoma with optical in vivo imaging technology. Methods： The murine B16 malignant melanoma cells,transferred with firefly luciferase gene by lentiviral vector（ B16-Luc）,were used as target cells. The cellular proliferation was detected with MTT colorimetric assay,the morphological observation and Annexin V and Propidium Iodide（ Annexin V / PI） double-labeling were employed to assess the cell apoptosis in vitro. The B16-Luc cells were implanted into the right front armpit of BALB / c mouse to establish the subcutaneously transplanted fluorescent malignant melanoma model in mice,the tumor-bearing mice were treated with 4 mg /（ kg·d） and 8 mg /（ kg·d） As2O3 by introperitoneal injection once a day for 25 days,respectively,the optical in vivo imaging system was used to continuously and dynamically monitor the tumor growth. At the end of the treatment the animals were sacrificed,and the tumor tissue was removed,the tumor tissue sections were prepared with HE staining and CD34 immunocytochemistry staining to examine necrosis and angiogenesis in tumors. Results： As2O3（ 1. 0 to 32. 0μmol /L） significantly inhibited the proliferation of B16-Luc cells and enforced the As2O3-treated cells to apoptosis in vitro,and As2O3 markedly reduced the growth of B16 malignant melanoma in vivo at a time-and dose-dependent manner. After As2O3 administration,the pathological and immunocytochemical measurement revealed that the numbers of microvessels in tumor tissue significantly decreased,and at the central region of tumor tissue where there existed extensive necrosis. Conclusions： Arsenic trioxide significantly inhibits the proliferation of murine B16 malignant melanoma cells via apoptosis-induction in vitro,and plays a role in anti-melanoma action on the transplanted malignant melanoma in vivo via repressing neovascularization to accelerate the necrosis of the tumor.