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目的研究经绿色荧光蛋白(GFP)基因修饰的人脉络膜黑色素瘤细胞株OCM-1-gfp在小鼠体内的生长过程、转移规律,以及可能影响肿瘤生长和转移的因素。方法用脂质体将GFP基因导入人脉络膜黑色素瘤细胞OCM-1,建立稳定、高水平表达GFP的克隆;分别接种到Balb/c裸鼠视网膜下和后大腿皮下,建立原位和异位肿瘤模型。眼内肿瘤生长情况用荧光显微镜直接观察,皮下肿瘤大小用游标卡尺测量;采用免疫组织化学方法对肿瘤内13种基因的表达进行检测。结果稳定表达GFP的脉络膜黑色素瘤细胞OCM-1-gfp基本保持了亲代细胞的特征;能在裸鼠体内形成肿瘤并继续生长和转移;在基因表达方面,肿瘤抑制基因p16染色呈阴性,p53染色呈强阳性。其他的肿瘤抑制基因:视网膜母细胞瘤易感基因(Rb)、p21,转录调控因子(E2F)、核因子κB(NFκB),细胞增生相关基因细胞周期素D1(cyclin D1)、增生细胞核抗原(PCNA),细胞凋亡相关基因bcl-2、bcl-XL/S和bax,以及表皮生长因子(EGF)及其受体(EGFR)呈现不同程度的阳性表达。结论GFP为直接观察脉络膜黑色素瘤在体内的生长和转移提供了标记;脉络膜黑色素瘤原位与异位移植瘤模型在成瘤率和生长情况方面无明显差异;由OCM-1-gfp生长形成的肿瘤p16、p53及NFκB、cyclin D1、PCNA及EGF和EGFR等多种基因表达异常。
Objective To study the growth and metastasis of human choroidal melanoma cell line OCM-1-gfp modified by green fluorescent protein (GFP) gene in mice and the factors that may affect tumor growth and metastasis. Methods GFP gene was transfected into human choroidal melanoma cell line OCM-1 by lipofectamine to establish stable and high-expression GFP-expressing clones. The GFP gene was inoculated subcutaneously in the subretinal and posterior thighs of Balb / c nude mice and established in situ and ectopic tumors model. Intraocular tumor growth was observed directly with a fluorescence microscope and the size of the subcutaneous tumor was measured with a vernier caliper. The expression of 13 genes in the tumor was detected by immunohistochemistry. Results OCM-1-gfp of choroidal melanoma cells stably expressing GFP basically maintained the characteristics of the parental cells, formed tumors in nude mice and continued to grow and metastasize, and in terms of gene expression, tumor suppressor gene p16 was negative and p53 staining Was strongly positive. Other tumor suppressor genes: Rb, p21, E2F, NFκB, cyclin D1, and proliferating cell nuclear antigen ( PCNA, bcl-2, bcl-XL / S and bax, as well as epidermal growth factor (EGF) and its receptor (EGFR) Conclusions GFP provides a direct indication of the growth and metastasis of choroidal melanoma in vivo. Choroidal melanoma in situ and in situ xenograft models have no significant difference in tumorigenicity and growth; the growth of OCM-1-gfp Tumor p16, p53 and NFκB, cyclin D1, PCNA and EGF and EGFR and other gene abnormalities.