目的： 以携带绿色荧光蛋白基因（eGFP）的重组逆转录病毒（RV）标记肿瘤细胞，建立小鼠体内长期、系统肿瘤细胞研究模型，并初步探讨其应用。方法： 制备高滴度eGFP-RV，感染小鼠白血病P388细胞，有限稀释法获得eGFP＋的单细胞克隆；在体外和DBA/2小鼠体内，分别以流式细胞术、荧光倒置显微镜、冰冻切片和普通病理切片、半固体集落培养和PCR等方法，观察小鼠体内eGFP标记肿瘤细胞的效率、定位和时效。结果 ： eGFP基因导入率达80.2%，经克隆筛选后eGFP＋率>99.2%。P388-eGFP细胞体外传代3个月后eGFP＋率为95.2%，体内序贯接种(56.3±1.25)d后eGFP+率为(93.3±0.50)% (n=3)。eGFP基因导入未影响P388细胞的体内外生物学特性。多聚甲醛灌注固定法和液氮速冻法可有效保存组织中的荧光，灌注固定法制备的标本适用于多种病理和组化染色观察；外周血、肝、脾、脑等脏器中eGFP＋细胞的分布、增殖，可用流式细胞仪和荧光显微镜等动态、定量观察，并可通过外周血的荧光细胞变化动态观察肿瘤细胞对治疗的反应；无菌获得的eGFP＋细胞可用于体外培养或体内序贯移植；PCR等技术可提高微量肿瘤细胞检测的敏感性。结论： 成功建立的模型可在体内外以多种方法长期、动态、敏感地追踪观察荧光标记的肿瘤细胞，可广泛应用于肿瘤和细胞组织工程等领域。

Objective: To establish aneGFP-gene-marked tumor cells model in murine for long-term in vivo research and to study its preliminary application.Methods:High titer eGFP retrovirus (RV) was prepared and was used to transfect mouse leukemia cells—P388 cells. P388-eGFP clones were obtained by limited dilution method. Wild type P388 (wtP388) was used as control; DBA/2 mice were inoculated with P388-eGFP abdominally or intravenously (n=10). eGFP+ cells from abdominal cavities or bone marrow of the dyed mice were used to inoculate new mice. eGFP+ P388 cells were studied in different tissues by Flow cytometry (FCM), semi-solid culture, fluorescence microscope, light microscope and PCR, etc.Results: It was found that 80.2% P388 cells were eGFP+ 48 h after retrovirus transfection. P388-eGFP clone selection increased the eGFP+ rate to 99.2%, and the rate remained at 95.2% after 3 months′ passaging. After inoculation in abdominal cavities for (56.3±1.25) d, the eGFP+ rate of P88-eGFP cells increased to (93.3±0.50)% (n=3). eGFP gene marking had no influence on the bioactivity of P388 cells. Paraformaldehydum and liquid nitrogen both effectively protected the fluorescence signals in the tissues. Perfusion fixation method was suitable for various kinds of pathological observation approach. FCM could be used to observe eGFP+ cells in the bone marrow, liver, spleen, thymus, peripheral blood, ascitic fluid，etc. The dynamic changes of eGFP+ cells could reflect the response of tumor cells to treatment. eGFP+ cells could be used for in vitro culture and in vivo transplantation. PCR is sensitive in detecting eGFP+ tumor cells in tissues. Conclusion: The present model allows for long term, dynamic and sensitive tracing of fluorescence-labeled cells in vivo, and can be widely used in tumor and cell engineering research.

国家自然科学基金（30172347）；上海市卫生系统百名跨世纪优秀学科带头人培养计划基金（98 BR029）