基于耐药特性联合单细胞培养识别肝癌干细胞克隆亚群  

Identification of liver cancer stem cells-containing cell subgroups based on resistance characteristics and unicellular culture

作　　者：陈娟[1] 区泳芳[1] 蔡捷[1] 陶璐[1] 陈相宜[2] 邝晓聪[1] 

机构地区：[1]广西医科大学病理与病理生理教研室,广西壮族自治区南宁市530021 [2]广西医科大学生理教研室,广西壮族自治区南宁市530021

出　　处：《世界华人消化杂志》2014年第3期319-326,共8页World Chinese Journal of Digestology

基　　金：广西自然科学基金资助项目;Nos.桂科自0991139;2013GXNSFAAO19244;广西科学研究与技术开发计划基金资助项目;No.桂科攻0993003C-1~~

摘　　要：目的:基于癌干细胞的耐药特性联合单细胞培养方法识别肝癌干细胞所在的克隆亚群.方法:以BEL-7404细胞系为研究对象,裸鼠体内移植瘤形成实验,并予阿霉素(adriamycin,ADM)8 mg/kg干预,待肿瘤直径为1.5 cm时取移植瘤细胞做原代培养,并在裸鼠体内连续传代4代,并将第4代移植瘤细胞命名为"BEL-7404-ADM-P4",根据成瘤时间检测肝癌干细胞富集情况.联合单细胞培养,取BEL-7404及BEL-7404-ADM-P4所形成的克隆根据克隆形态分为全克隆、部分克隆、旁克隆,检测BEL-7404及BEL-7404-ADM-P4单克隆中全克隆、部分克隆、旁克隆的癌干特性,即增殖能力、克隆形成率及悬浮球形成率;取其全克隆、部分克隆、旁克隆做hoechst33342染色,并在共聚焦显微镜下观察其染色情况;根据BEL-7404及BEL-7404-ADM-P4单克隆中全克隆、部分克隆、旁克隆的增殖能力、克隆形成率及悬浮球形成率以及hoechst33342染色情况识别癌干细胞所在的克隆亚群.结果:裸鼠体内低剂量阿霉素干预,在体内连续成瘤传代过程中,裸鼠皮下移植瘤成瘤率均为100%,且成瘤时间从第一代到第四代均有所缩短,以此达到了肝癌干细胞的初步富集;增殖能力:全克隆增殖速度最快,细胞量最大,部分克隆次之,旁克隆增殖速度最慢,并于第10天开始出现细胞皱缩死亡;克隆形成率:BEL-7404-ADM-P4-H高于BEL-7404及BEL-7404-ADM-P4-M,差异有统计学意义(P<0.05);悬浮球形成率:仅BEL-7404-H及BEL-7404-ADM-P4-H可形成悬浮球,其余细胞系不形成悬浮球,而BEL-7404-H及BEL-7404-ADM-P4-H悬浮球形成率比较,无统计学意义(P>0.05);BEL-7404及BEL-7404-ADM-P4单克隆中全克隆、部分克隆、旁克隆hoechst33342染色情况:BEL-7404及BEL-7404-A D M-P4单克隆中全克隆都有极少数不染和低染的细胞存在,但部分克隆、旁克隆全染,且BEL-7404单克隆中全克隆、部分克隆、旁克隆hoechst33342荧光强度均强于BEL-7404-ADM-P4单克隆中全�AIM： To identify liver cancer stem cells-contain- ing cell subgroups based on resistance character- istics and unicellular culture.ed subcutaneously in nude mice to induce tumor formation, and adriamycin （8 mg/kg） interven- tion was given. When the tumor diameter was 1.5 cm, tumor tissues were collected for primary culture. The cells were inoculated subcutane- ously in nude mice again. After four consecutive generations in nude mice, the tumor cells were named ＂BEL-7404-ADM-P4＂. Based on the tu- mor forming time, the enrichment of liver cancer stem cells was tested. Using unicellular culture, clones from BEL-7404 and BEL-7404-ADM-P4 cells were divided into holoclones, meroclones and pareclones to test the cancer stem cell char- acteristics （renewal ability, clone formation and sphere formation）. Holoclones, meroclones and pareclones were stained with hoechst33342 and observed under a confocal microscope. Based on the renewal ability, clone formation rate and sphere formation rate and hoechst33342 stain- ing properties, cancer stem cells subpopulations were identified. RESULTS： The tumor formation rate was 100%, and tumor formation time was shortened from the first generation to the fourth generation, which suggested the preliminary enrichment of liver cancer stem cells. Holoclones showed the fastest growth and the largest cell volume, fol- lowed by meroclones and pareclones. On the 10th day, pareclones started to shrivel and die. The clone formation rate of BEL-7404-ADM- P4-H cells was significantly higher than those of BEL-7404 and BEL-7404-ADM-P4-M cells （P 〈 0.05）. Only BEL-7404-H and BEL-7404-ADM- P4-H cells could form spheres, and there was no significant difference in sphere formation rate between BEL-7404-H and BEL-7404-ADM-P4-H cells. In BEL-7404 and BEL-7404-ADM-P4 mono- clones and holoclones, there were a small num- ber of lowly hoechst33342 stained or non-stained cells, but cells in meroclones and pareclone cells were all stained. The hoechst33342 fluorescence intensity of BEL-740

关 键 词：阿霉素干预 肝癌干细胞 单细胞培养 全克隆 部分克隆 旁克隆 

分 类 号：R735.7[医药卫生—肿瘤]