人肝癌高增殖率细胞筛选及致瘤特性分析  

Screening of human liver cancer cells with high growth rate and analysis of their tumorigenic characteristics

作　　者：潘兴华[1] 魏洪林 郭中敏[3] 庞荣清[1] 蔡学敏[1] 

机构地区：[1]成都军区昆明总医院干细胞与组织器官工程研究中心,昆明650032 [2]昆明总医院曲靖医疗所 [3]中山大学医学实验动物中心

出　　处：《西南国防医药》2010年第10期1045-1048,F0004,共5页Medical Journal of National Defending Forces in Southwest China

基　　金：国家自然科学基金(30270647);中国博士后基金(2003033431)

摘　　要：目的分离培养人肝癌组织中的高增殖率细胞并观察其生物学特性。方法无菌采集人肝细胞癌组织,切成1mm×1mm×1mm组织块并置于裸鼠肾包膜下生长3w;相同方法反复体内传代3次后,将组织块剪碎,胶原酶消化分离为单细胞悬液,接种于培养瓶中,用阿霉素(Adm)5mg/L处理72h获得抗药细胞。通过体外培养和体内生长观察该细胞的生长规律;用免疫细胞化学染色观察其表达C-kit、CD133和甲胎蛋白(AFP)的情况。结果原代肝癌细胞直接分离培养生长较缓慢,经体内传代3次后,分离培养获得生长良好细胞,再经抗肿瘤药物阿霉素共培养后,获得耐Adm细胞。经体内生长特性观察发现,Adm筛选后,相同数量细胞的致瘤生长速度升高。经免疫组化染色分析发现,筛选后的细胞表达C-kit、AFP、CD133。结论通过组织块体内传代、体外抗癌药物筛选,初步获得表达肝干细胞标志性抗原的高致瘤性细胞。Objective To isolate and culture the cancer cells with high growth rate from human hepatoma tissues, and to observe their biologic characteristics. Methods Human hepatoma tissues were collected under sterile conditions, and then cut into 1 mm × 1 mm × 1 mm of tissue blocks that were subsequently placed under the renal envelopes of nude mice for 3 w. After three intracorporal passages of liver cancer cells with the same method, the cancer tissues were taken out from the animals and cut into very small pieces that were digested and isolated as monocelluar suspension by the collagenase. Then these cells were inoculated into culture flasks, and drug resistant cancer cells were obtained after treatment with 5 mg/L of adriamycin （Adm）for 72 h. Their growth regularity was observed when they were cultured in vitro and grew subcutaneously in mice, and then the expression of C - kit, CD133 and AFP in these cells was measured by immunohistoehemical staining. Results Primary liver cancer cells appeared to grow slowly after isolated and cultured directly. However, they grew well following three generations of passage in vivo. Having been eoeuhured with antitumor drug Adm, the cancer cells against it were obtained. The observation of growth characteristics demonstrated that the same amount of cancer cells could induce a faster growth of tumor after screening by Adm than those without screening. Moreover, the immunohistochemieal staining denmnstrated that C - kit,AFP and CD133 were expressed in cells screened by Adm. Conclusion Cells with high oncogenieity expressing marker antigens of hepatic stem cells are obtained through Adm screening in vitro after the passage of human liver cancer cells in vivo using tissue block method.

关 键 词：肝癌 高增殖细胞 致瘤性 

分 类 号：R735.7[医药卫生—肿瘤]