摘要
目的建立胆囊癌肝转移模型并通过模型筛选高转移亚群细胞,为胆囊癌肝转移研究提供良好的实验工具。方法胆囊癌GBC-SD细胞在含10％胎牛血清的RPMI1645体外培养、胰蛋白酶分离、生理盐水制备细胞悬液1×107／ml,4-6周龄裸小鼠脾脏接种建立胆囊癌转移模型;从实验动物肝转移灶中分离肿瘤细胞,经过原代、传代培养,同样的方法建立转移模型,进行第二轮和第三轮高转移表型胆囊癌细胞亚群的筛选。光镜下观察转移灶和筛选细胞的形态特征;抽提细胞与实验动物肝组织DNA,PCR扩增三对人类特异的微卫星序列。结果建立了裸小鼠胆囊癌肝脏转移模型,转移灶分布肝实质各处,但以边缘为主。左叶多受侵犯,部分全肝转移。组织学观察转移病灶为腺癌。第一轮、第二轮、第三轮出转移模型在接种后10周、7周、5周形成肉眼转移灶,转移率为60％、70％、90％。从第三轮转移灶中筛选出高转移胆囊癌细胞亚群,命名为GBC-SD／M3。与亲代细胞GBC-SD相比,所筛选的GBC-SD／M3具有相似的形态特征。GBC-SD、GBC-SD／M3在微卫星D14S68、D18S69、D20S199位点扩增产物片段完全相同,荷瘤裸鼠肝脏没有扩增出相应的产物片段。结论本实验成功建立了胆囊癌肝转移模型;从转移模型中筛选出高转移胆囊癌细胞亚群GBC-SD／M3,与亲代细胞GBC-SD相比,形态和遗传背景保留一致性,但具有更高转移能力。
Objective To establish an experimental liver metastatic model of gallbladder cancer and isolate the subpopulation with high metastatic potential from the model, which may serve as a reliable tool in research on liver metastasis of gallbladder cancer in vivo and in vitro. Methods Human gallbladder cancer cells of the line GBC-SD were cultured. Ten nude athymic BALB/c mice, aged 4 ～ 5 weeks, underwent inoculation of suspension of GBC-SD cells into the spleens and then their spleens were resected. Three weeks later laparotomy was performed to observe if liver metastasis occurred. Once liver metastasis was discovered, the mice were killed and the livers were taken out to undergo microscopy, tumor cells were isolated from the metastatic foci and cultured in vitro, and then inoculated into other 10 mice in the same way as their parents cells for the second round of selection. The similar steps were repeated, altogether for three rounds of selection and a total 30 mice were inoculated in 3 rounds so as to find subpopulation with high metastatic potential. Another 10 mice underwent subcutaneous inoculation, 90 days later the mice were killed to observe if pulmonary metastasis occurred. PCR was used to detect the 3 microsatellite sequences D14S68, D18S69, and D20S199 in the DNA samples of the gallbladder cancer cells of the parent cell line GBC-SD, the isolated cells of the subpopulation with high metastatic potential, and the liver of experimental animal. Results 90% of the mice inoculated subcutaneously with the GBC-SD cells developed subcutaneous tumors, however, no mouse in this group died and no pulmonary metastasis was found. The liver metastatic rate was 65% in the 10 mice undergoing intrasplenic inoculation. Thus a metastatic model of human gallbladder cancer in nude mice was established. The liver metastatic tumors were uniformly distributed throughout the liver parenchyma with predominance to the periphery. Despite multiple sites of involvement,the left lobes were most commonly affected in all experimental animals. Histological examination of the metastatic lesion demonstrated adenocarcinoma. Gross hepatic metastasis was detected 10, 7, and 5 weeks after the inoculation respectively in the first, second, and third round selection respectively with an incidence rate of metastases of 60% , 70% , and 90% respectively. From the third round metastatic model a subpopulation with high metastatic potential was isolated and designated as GBC-SD/M, which exhibited the similar histological characteristics as its parent cell line GBC-SD under inverted light microscopy. The three amplified bands at the sites 14S68, D18S69, and D20S199, amplified with the three pairs of specific primers for the three homo-specific microsatellites, were detected in the GBC-SD cells and GBC-SD/M cells, but not in the liver of tumor-bearing animal. Conclusion A liver metastasis model of human gallbladder cancer has been established, a reliable and efficient tool for study on the metastasis of gallbladder cancer to liver in vivo. Isolated from hepatic metastasis, the line of GBC-SD/M is a subpopulation with high metastatic potential, retaining the histological properties and identification of genetic background of its parent cell line GBC-SD.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2006年第30期2117-2121,共5页
National Medical Journal of China
基金
国家自然科学基金资助项目(30571831)
关键词
胆囊肿瘤
肿瘤转移
动物
实验
Gallbladder neoplasm
Neoplasm metastasis
Diseases model, animal
