CD133+细胞的干性鉴定及131I-CD133抗体对其体内外抑制作用

Identification of the stem property of CD133+ liver cancer cells and inhibition effect of 131I-labeled anti-CD133 mAb on them in vitro and vivo

摘要:

目的：研究CD133+细胞的干性鉴定和131I-CD133抗体在体内外对人肝癌CD133 -HepG2干细胞的抑制作用。方法：氯胺T法制备并鉴定131I-CD133抗体；免疫磁珠（magnetic-activated cell sorting，MACS）分选CD133 -HepG2细胞；流式细胞仪（flow cytometry，FCM）检测分选前后CD133表达率；体外克隆形成实验、成球实验和体内成瘤实验验证其干细胞特性；将分选出的CD133 细胞分组为CD133抗体、131I、131I-CD133抗体和131I CD133抗体 4个组，MTT法检测不同处理后不同组中CD133 细胞生长抑制率；成功构建人肝癌CD133 -HepG2移植瘤模型；随机分4组，1次/2 d给予尾静脉用药，共14次。4周后，处死小鼠，比较肿瘤的体积、质量、计算抑瘤率；HE染色观察肿瘤组织病理学改变。结果：131I-CD133抗体标记率为89.34%，放化纯度为98.21%。流式显示分选前后CD133表达率分别为（1.78±0.54）%和（98.46±0.97）%。成球实验、克隆形成实验和裸鼠成瘤实验显现CD133 细胞相对于CD133-细胞更具有干细胞特性。131I-CD133抗体治疗组体外对细胞抑制率及体内抑瘤率明显高于其余各实验组，差异具有统计学意义（P<0.05）。结论：131I-CD133抗体在体内外均能有效抑制人肝癌CD133 -HepG2细胞的生长。

Abstract:

Objective：To study the identification the stem property of CD133+ liver cancer cells and inhibition effect of 131I-labeled monoclonal antibody（mAb）against CD133 on them in vitro and vivo. Methods：The monoclonal antibody CD133 labeled with 131I was prepared using the chloramines-T method and its stability was evaluated. Magnetic-activated cell sorting（MACS） was used to isolate CD133 and CD133- cells from HepG2 cells. Flow cytometry（FCM） was used to detect the expression of CD133 before and after the cell isolation. The stem cell properties of sorted CD133 cells were validated by sphere-forming assay，colony formation assay in vitro and tumorigenesis experiment in vivo. There were four groups including 131I-labeled anti-CD133 mAb group，131I group，CD133 mAb group and 131I CD133 mAb group. The inhibitory effects of different treatments on the proliferation of CD133 cells were measured by MTT assy. The animal model was established by subcutaneous inoculation of CD133 -HepG2 cells（5×104） to right front legs of BALB/c mice. After tumor model being established，12 mice were randomly divided into 4 groups. Drugs were injected into the tail vein of the nude mice at a frequency of one time/2 d（14 times in total）. The tumor size and volume were measured twice a week after the treatment and the tumor inhibitory rate was calculated. After 4 weeks，the mice were sacrificed for pathological examination. Results：The labeling ratio of the 131I-labeled anti-CD133 mAb was 89.34% and the radiochemical purity was 98.21%. The sorted CD133 cells showed a high purity（98.46±0.97）% compared with that before cell sorting（1.78±0.54）%. CD133 cells showed a high tumor sphere formation ability and turmorigenesis capacity compared with those of CD133- cells. The tumor inhibitory rate of CD133 cells was significantly higher in 131I-labeled anti-CD133 mAb group than in 131I group，CD133 mAb group and blank control group in vivo and vitro（P<0.05）. Conclusion：１31I-labeled anti-CD133 mAb can effectively inhibit the growth of CD133 -HepG2 cells in vivo and vitro.