绿色荧光蛋白标记的脉络膜恶性黑色素瘤原位移植瘤模型

目的 建立新型裸鼠脉络膜恶性黑色素 瘤原位移植瘤模型。 方法 利用脂质体将绿色荧光蛋白（GFP）真核表达 质粒pEGFP-N₁转入人脉络膜恶性黑色素瘤细胞（OC-1），新霉素、荧光显微镜及流式细胞仪筛选稳定表达GFP的细胞克隆。将2 μl细胞浓度为4.5×10⁷~5.5×10⁷个/ml的细胞悬液注射到麻醉后的40只裸鼠右眼视网膜下间隙，左眼不注射作为对照眼。手术后利用带荧光的体视显微镜连续观察肿瘤生长情况，分别于肿瘤生长的眼内期、眼外期及衰竭期处死动物，荧光显微镜观察裸鼠视神经、颅底、肺、肝、肾的肿瘤转移情况，并行肿瘤组织的GFP免疫组织化学染色。 结果 手术后10~12 d肿瘤开始生长，血管扩张、扭曲，新生血管形成；手术后20~22 d肿瘤占满玻璃体腔；手术后24~26 d肿瘤生长至眼外；眼外期后，迅速进入衰竭期，衰竭期嗅球、肾、肺、肝脏有转移灶。移植瘤病理组织学表现类似于人脉络膜恶性黑色素瘤；免疫组织化学染色肿瘤细胞GFP染色阳性。 结论 以GFP基因标记的OCM-1经裸鼠视网膜下间隙移植建立的脉络膜恶性黑色素瘤原位移植瘤模型，为研究自然状态下肿瘤的生长和转移提供了一个新的方法。 （中华眼底病杂志，2004，20：245-248）

Model of orthotopic transplatation tumor of malignant choroidal melanoma labeled by green fluorescent protein

Objective To establish a new model of orthotopic-transplatation tumor of human malignant choroidal melanoma. Methods pEGFP-N₁, the eukaryotic expressive plasmid of green fluorescent protein (GFP), was transfered into human malignant choroidal melanoma cell line (OCM-1) by liposome lipofectanine, then the cell clones with stable GFP expression were screened out by means of neomycin, fluorescence microscope, and flow cytometer. Two μl cell suspension of OCM-1 cells with GFP expression with the density of 4.5×10⁷-5.5×10⁷ cells/ml was injected into the subretinal space of right eyes of 40 nude mice (40 eyes) under binocular operating microscope with left eyes as the control ones. The growth of the transplanted malignant choroidal melanoma was observed in vivo using the fluorescence stereomicroscope. The mice were killed at different time after the operation to observe the metastasis of the tumor to optic nerve, brain and other organs including lung, liver, kidney and spleen. Moreover, pathological detection and immunohistochemical staining of GFP was carried out. Results At the postoperative 10th-12th days, the growths of the transplanted malignant choroidal melanoma with dilated and distorted blood vessels and neovascularization were observed; at the postope rative 20th-22nd days, the melanoma occupied the whole cavity of vitreous body; and at the postoperative 24th-26th days, the transplanted tumor grew out of the eye. Metastases of the carcinoma to olfactory bulb, kidney, lung and liver were seen at the failure phase soon after the extra-ocular phase. The histopathological characteristics of the transplanted tumor were similar to those of human, and the results of immunohistochemical staining showed positive expression of GFP in the tumor cells. Conclusion The orthotopic model of malignant choroidal melanoma set up via injection of human malignant choroidal melanoma cells labeled by GFP into the subretinal space of nude mice may provide a new approach to investigate the natural courses of growth and metastasis of malignant choroidal melanoma. (Chin J Ocul Fundus Dis，2004，20：245-248)