CAJ | 학술논문

目的探讨采用一种新型高效的肝癌干细胞分离方法,并对获得的肝癌干细胞进行鉴定.方法应用无血清肿瘤干细胞培养基,悬浮培养SMMC-7721形成克隆,将悬浮细胞收集后裸鼠皮下接种,并予顺铂干预,取耐药细胞继续悬浮培养获得克隆,分别采用定量PCR、免疫荧光法和流式细胞法检测干细胞和肿瘤干细胞标志的表达情况.结果在肿瘤干细胞培养基和顺铂的双重筛选下,SMMC-7721能有效形成悬浮生长的细胞克隆.检测结果提示,与普通肝癌细胞相比,获得的干细胞克隆高表达多种干细胞标志NANOG、OCT-4、SOX-2、Notch和肿瘤干细胞标志CD24(90.0％)、CD133(6.1％)、CD90(4.8％),其中尤以CD24最为明显,差异均有统计学意义(均P＜0.01).结论无血清体外培养+体内增殖+耐药筛选相结合是一种能有效分离纯化肿瘤干细胞的新方法,可从SMMC-7721中分离出具有干细胞特征并高表达CD24的肝癌干细胞克隆.
목적 탐토채용일충신형고효적간암간세포분리방법,병대획득적간암간세포진행감정.방법 응용무혈청종류간세포배양기,현부배양SMMC-7721형성극륭,장현부세포수집후라서피하접충,병여순박간예,취내약세포계속현부배양획득극륭,분별채용정량PCR、면역형광법화류식세포법검측간세포화종류간세포표지적표체정황.결과 재종류간세포배양기화순박적쌍중사선하,SMMC-7721능유효형성현부생장적세포극륭.검측결과제시,여보통간암세포상비,획득적간세포극륭고표체다충간세포표지NANOG、OCT-4、SOX-2、Notch화종류간세포표지CD24(90.0％)、CD133(6.1％)、CD90(4.8％),기중우이CD24최위명현,차이균유통계학의의(균P＜0.01).결론 무혈청체외배양+체내증식+내약사선상결합시일충능유효분리순화종류간세포적신방법,가종SMMC-7721중분리출구유간세포특정병고표체CD24적간암간세포극륭.
Objective To explore a new efficient purification method of hepatocellular carcinoma (HCC) stem cells and identify their features.Methods Human hepatocellular carcinoma cell line SMMC-7721 was cultured in sphere-culture system of polyhema-treated dish and tumor stem cell specific medium.Upon the formation of cellular sphere,the cells were inoculated subcutaneously into immunocompromised mice and received the interventions of different concentrations of cisplatin.Then the drug-resistant cells were purified and re-cultured in TSC medium.Finally the stem cell markers and tumor stem cell markers were determined through real-time polymerase chain reaction (PCR),immunofluorescence method and flow cytometry.Results Through the double filter of TSC medium and cisplatin-resistance,SMMC-7721 stem cells could be grown in a suspended form and formed spheres in TSC medium.The stem cell markers (NANOG,OCT-4,SOX-2 and Notch) and tumor stem cell markers (CD24,90.0％ ; CD133,6.1％ ;CD90,4.8％) were all over-expressed in purified cancer stem cells as compared with ordinary cells.And the over-expression of CD24 was the most obvious.Conclusions The combination of in vitro cell culture with TSC medium,in vivo proliferation and cisplatin resistance test is a new efficient method of purifying hepatocellular carcinoma stem cells.Tumor stem cell with stem cell characteristics and an over-expression of CD24 may be cloned from SMMC-7721.