中文摘要：

目的分离、鉴定CD133^＋人肝癌干细胞以及^131I-CD133mAb对其生物学效应的影响。方法为选择最适的细胞系，流式细胞仪检测Huh-7和HepG2细胞中CD133表达率；磁珠分选Huh-7细胞，流式细胞仪检测分选后CD133表达率；体外成球、克隆形成实验及体内成瘤实验验证干细胞特性；氯胺T法制备^131I标记CD133单克隆抗体（^131I—CD133mAb）并鉴定；取分选后的CD133＋-Huh-7细胞、Huh-7细胞和HepG2细胞进行实验，将每种细胞分成4组（^131I—CD133mAb组、131I组、CD133mAb组、131I＋CD133mAb组）；MTT法检测各组对CD133＋-Huh-7细胞生长抑制的最适剂量和各组在最适剂量作用后24、48、72h对3组细胞生长抑制率；流式细胞仪检测^131I-CD133mAb作用3组细胞72h时细胞凋亡和细胞周期的变化。结果Huh-7和HepG2细胞系CD133表达率分别为18．8％和5．2％，故选用Huh-7细胞进行分选；磁珠分选后CD133＋-Huh-7细胞CD133的表达率为99．28％，与分选前比较差异有统计学意义（P〈0．01）。CD133＋-Huh-7细胞相对于CD133--Huh-7细胞具有更强的体外成球能力、克隆形成能力和体内成瘤能力。^131I-CD133mAb标记率为87．92％，放射化学纯度为97．54％，具有较好的稳定性和免疫活性。当^131I 4．8MBq／100μL、CD133mAb4．8μg／100μL时对CD133＋-Huh-7细胞抑制率最高（P〈0．05），取此放射性浓度进行实验。24、48、72h时各组对CD133＋-Huh-7细胞的抑制作用明显高于Huh-7和HepG2细胞组，^131I-CD133mAb组对3种细胞生长抑制率明显高于其余各组，抑制率随时间的增加而升高（P〈0．01）。^131I-CD133mAb作用CD133＋-Huh-7细胞72h后凋亡率为31．21％，明显高于其余2组（P〈0．05）。细胞周期检测结果显示CD133＋-Huh-7细胞G0／G1期减少54．77％，明显高于其余2组（P〈0．05）。结论CD133＋细胞具有肿瘤干细胞特性，^131I-CD133mAb能特异性结合CD133＋-Huh-7细胞，调控细胞?

英文摘要：

Objective To study the sorting of CD133 ＋ liver cancer cells having the features of cancer stem cells, and to study 131I-labeled anti-CD133 monoclonal antibody （1311-CD133mAb） biological effects on the cells. Methods Flow cytometry （FCM） was used to detect the CD133 expression on Huh-7 cells and HepG2 cells. Magnetic-activated cell sorting （MACS） was used to isolate CD133 ＋ and CD133- cells from Huh-7 cells. FCM was used to detect the expression of CD133 after cell isolation. The stem cell properties of sorted CD133 ＋ cells were validated by sphere-forming assay and colony formation assay in vitro and BALB/c mice transplantation tumor experiments in vivo. 131I_CD133mAb was prepared by the chloramines-T method and then identified. CD133 ＋ Huh-7 cells, Huh-7 cells and HepG2 cells were separately divided into 4 groups inclu- ding 131I-CD133mAb group,131I group, CD133mAb group andl31I ＋ CD133mAb group. The optimal inhibitory concentration and the inhibitory effects at 24, 48, and 72 h on cell proliferation in each group were measured by MTI＂ assay. The apoptosis and cell cycle change rates of the three cells 72 h after 131I-CD133mAb treatment were evaluated by FCM. Results CD133 expression rates on Huh-7 cells, HepG2 cells and CD133＋ Huh-7 cells were 18.8%, 5.2% and 99.28%, respectively. CD133 ＋ Huh-7 cells showed a higher tumor sphere formation ability and tumor-genesis capacity compared with CD133- Huh-7 cells. The labeling ratio of 131I- CD133mAb was 87.92% and the radiochemical-purity was 97.54%. The growth inhibition rate on CD133 ＋ Huh-7 cells was the highest when 131I was 4. 8 MBq/100 ~L and CD133mAb was 4.8 μg/100μL as shown by MTF assay （ P 〈 0.05 ）. In all groups at 24, 48, 72 hours after treatment, the growth inhibitory rate on CD133 ＋ Huh-7 cells was significantly higher than that on Huh-7 cells and HepG2 cells and the growth inhibitory rate of 1311-CD133mAb group was obviously the highest as proven by MTr assay, which showed a time-dependent manner （P 〈0. 05）