首株来源于转移灶的人胆囊癌细胞系EH-GB1的建立及鉴定

Establishment and characterization of a human gallbladder carcinoma cell line EH-GB1 originated from a metastatic tumor

目的建立转移性胆囊癌细胞系，鉴定并研究其生物学特性。方法从人胆囊癌转移灶取材，将标本分离成单细胞悬液，用含15％胎牛血清的DMEM培养液进行原代和传代培养，培养成功后命名为EH-GB1细胞。电镜下观察EH—GB1细胞的形态，绘制细胞生长曲线。SCID小鼠皮下接种EH-GB1细胞，观察EH—GB1细胞的成瘤能力。采用免疫组化法，检测小鼠移植瘤组织中CA19-9的表达。分别用携带绿色荧光蛋白（GFP）和红色荧光蛋白报告基因（Red2）的慢病毒感染EH-GB1细胞，观察EH—GB1细胞表达GFP和Red2的情况。结果EH—GB1细胞体外连续传代20代以上，形态上具有典型的恶性上皮细胞特征，倍增时间约为24h。SCID小鼠皮下接种EH—GB1细胞后的成瘤率为100％。移植瘤细胞中CA19—9呈强阳性表达。EH—GB1细胞感染携带GFP或Red2基因的慢病毒后，可稳定地表达GFP和Red2。结论EH—GB1细胞有望成为首株来源于转移灶的稳定的人胆囊癌细胞系，转染并持续表达GFP和Red2的EH-GB1细胞是胆囊癌临床与基础研究中的理想实验模型。

Objective To establish a human gallbladder carcinoma ceil line derived from a metastatic gallbladder carcinoma and identify its biological characteristics. Methods Tissue samples were separated from the surgical specimen obtained from a patient with metastatic carcinoma and single-cell suspension was prepared. Then the cells were cultured in DMEM medium supplemented with 15% fetal bovine serum. The morphology of tumor cells was observed under an electron microscope. The cell growth curve was plotted. The tumorigenicity of the cell line was studied by subcutaneous inoculation in SCID mice. The cells were infected by lentiviral vector carrying fluorescent report genes （ lenti-GFP and lenti-Red2 ） separately for expressions of GFP and Red2, respectively. Results A novel metastatic gallbladder carcinoma cell line was successfully established and named ＂EH-GB1 ＂. It could be passaged for over 20 generations with typical malignant epithelial morphology and a stable growth cycle of 24 h. Tumors were formed in all of the 10 SCID mice inoculated with EH-GB1 cells subcutaneously, and the tumor ceils were tumor marker CA19-9-positive. Continuous expressions of fluorescent report genes were observed in EH-GB1 cells infected by lenti-GFP and lenti-Red2. Conclusion EH-GBI cells might be the first stable ceil line of human gallbladder carcinoma established from a metastatic focus of gallbladder carcinoma. This cell line with continuous expressions of GFP and Red2 might be a novel and perfect experimental model for clinical and basic research on gallbladder carcinoma.