用于活体成像的小鼠肝癌移植瘤模型的建立

摘要

Objective To establish xenograft mouse models for studying human hepatocellular carcinoma by using in vivo imaging system. Methods We first transfected pGL3 (LUC/NEO)plasmid into human liver cancer cells SMMC-7721, Huh-7, Hep3B, MHCC-97H, HCC-LM6, PLC/PRF/5 and screened cell lines to acquire stably expressing luciferase reporter gene with G418. Then we analyzed the correlation of luciferase activity and cell numbers according to in vitro bioluminescence and growth characteristics. We selected suitable clones and inoculated subcutaneously into nude mice to construct liver cancer xenograft models. Using an in vivo imaging system, we monitored the growth of the tumors. We verified the growth and metastasis of tumorgenesis by tissue sections with Hematoxylin and Eosin (HE) staining. Results We constructed six stably expressing luciferase reporter gene liver cancer cell lines and verified the activity of the luciferase in these cell lines in vitro. The experiment expressed that the stable cells of SMMC-7721 Luc+, Huh7Luc+, PLC/PRF/5Luc+, MHCC-97HLuc+ had the characteristics of tumor genesis. Using in vivo monitoring system, the growth of the stable cell lines SMMC-7721Luc+, MHCC-97HLuc+, HCC-LM6Luc+, PLC/PRF/5Luc+ injected into liver in situ could be monitored. And using in vivo monitoring system, the growth of the stably cell lines MHCC-97HLuc+, PLC/PRF/5Luc+ injected from the spleen could also be monitored. The HE staining showed that liver cancer cells existed in the liver of the nude mouse. Conclusion The research successfully establish xenograft mouse models and they provide convenient, sensitive, intuitive and stable tools for studying the mechanisms of liver cancer's growth-transference and the research of antitumor drugs.%目的 建立可用于活体成像的小鼠肝癌移植瘤模型.方法 利用脂质体将荧光素酶表达载体pGL3(LUC/NEO)转染至人肝癌细胞株SMMC-7721、Huh-7、Hep3B、MHCC-97H、HCC-LM6、PLC/PRF/5中,经G418筛选获得稳定表达荧光素酶的细胞克隆.根据体外生物发光情况及细胞的生长特性,从中挑选合适克隆,进行裸鼠皮下接种,建立肝癌移植瘤模型.利用活体成像系统监测肿瘤的生长转移情况,并对肝脏组织进行HE染色分析肿瘤细胞的生长分布特点.结果 建立了6株肝癌细胞株的荧光素酶基因稳定表达的亚克隆,并且体外验证了各种细胞株中的荧光素酶活性情况；裸鼠皮下成瘤实验显示了SMMC-7721Luc+、Huh7Luc+、PLC/PRF/5Luc+、MHCC-97HLuc+稳定细胞具有成瘤特性；裸鼠肝脏原位注射肝癌细胞(SMMC-7721Luc+、MHCC-97HLuc+、HCC-LM6Luc+、PLC/PRF/5Luc+),利用活体成像系统能够有效对活体肿瘤生长情况进行监测；裸鼠脾动脉注射肝癌细胞(MHCC-97HLuc+、PLC/PRF/5Luc+),利用活体成像系统能够监测到小鼠肝癌模型的生长状况,之后处死小鼠,对肝脏组织HE染色发现肝癌细胞局部分布于肝脏中.结论 本研究成功地构建了可用于活体成像的小鼠肝癌移植瘤模型,模型稳定可靠、直观、灵敏,为肿瘤生长转移机制的研究及抗肿瘤药物的研发提供了重要工具.