绿色荧光蛋白标记的裸鼠结肠癌荧光原位移植模型的建立及生物学特性

摘要

目的 建立稳定、高表达绿色荧光蛋白(GFP)标记的裸鼠结肠癌原位移植模型并观察其生物特性.方法 构建GFP pLPCX逆转录病毒质粒,将其转染至人结肠癌HCT116细胞,建立裸鼠的皮下瘤模型,活体荧光影像观察到绿色荧光标志的肿瘤生长至10 mm×10 mm,取肿瘤分别对15只BALB/C裸鼠进行结肠原位移植.利用荧光影像系统在不同时间点观察裸鼠原位移植瘤的生长、转移情况.不同时间点的卡尺与荧光测量结果分析采用t检验,组间比较采用重复测量设计的方差分析.结果 15只裸鼠结肠癌原位荧光移植模型均成功构建,未发生与手术相关的并发症和死亡.术后第3周,所有动物模型在荧光下观察到肿瘤.肿瘤体积随着时间推移逐步增大,术后3、4、5、6、7周不同时间点整体荧光影像测量原位移植肿瘤计算的体积＞体表游标卡尺测量计算的体积,但差异无统计学意义(f=-1.280,-1.115,-0.718,-0.199,-0.386,P＞0.05)；测量方式与时间的交互作用,两种测量方法的结果比较,差异有统计学意义(F =29.546,P＜0.05).实验终点时存活8只动物模型,其中6/8的动物模型发生肿瘤转移.结论 结肠癌荧光原位移植模型技术可行,能够进行体内实时及无创地动态观察和分析肿瘤细胞的生长与转移情况.%Objective To establish a stable orthotopic model with high green fluorescent protein (GFP) expression in nude mice and observe its biological features.Methods Human HCT116 colon cancer cells transfected with GFP pLPCX retroviral plasmid were used to build a subcutaneous tumor model in nude mice.Fifteen BALB/C nude mice were selected to underwent orthotopic transplantation of colon when the GFP-labeled tumor grew to 10 mm × 10 mm as observed by in vivo fluorescent microscopy.The growth and metastasis of orthotopically implanted colon cancer cells were observed with fluorescent imaging system at different time points.The differences of the tumor size measured by peripheral vernier caliper and fluorescent imaging system were analyzed using the t test,and the differences in different groups were analyzed using the analysis of variance.Results GFP-labeled colon cancer models were successfully established in all the 15 nude mice,and there was no surgery-related complications or death.Tumors marked by GFP were observed under fluoroscope in week 3.The size of the tumors progressively increased with time.The volumes of the orthotopically transplanted tumors obtained from global measurement using fluorescent imaging system were greater than those measured by peripheral vernier calipers at postoperative week 3,4,5,6,7,while no statistically significant difference was observed (t =-1.280,-1.115,-0.718,-0.199,-0.386,P ＞0.05).There was a significant difference in the interation of measure method and different time points (F =29.546,P ＜ 0.05).Eight nude mice survived at the end of the experiment,and tumor metastasis was observed in 6 mice.Conclusions It is technically feasible to construct GFP-labeled colon cancer orthotopic transplantation model.The mice model could be used for real-time,in vivo,non-invasive and dynamic observation and analysis of the growth and metastasis of tumor cells.