分离、鉴定CD133^+人肝癌干细胞以及^(131)I-CD133mAb对其生物学效应的影响

Isolation and identification of CD133-positive liver cancer stem cells and ^(131)I-CD133mAb biological effects on the cells

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(131ICD133mAb)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(131ICD133mAb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、72 h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72 h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 4.8 MBq/100μL、CD133mAb 4.8μg/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、72 h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72 h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,调控细胞周期和诱导细胞凋亡。

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）. The apoptotic rate of CD133μ Huh-7 cells in 72 h after 131I-CD133mAb treatment was 31.21% , significantly higher than that of other groups （P 〈0. 05）. The rate of CD133 μ Huh-7 cells in G0/G1 phase was reduced to 54.77%, significantly greater than that of other groups （ P 〈 0.05 ）. Gonclusion CD133 ＋ cells have the characteristics of cancer stem cells. 131I-CD133mAb can effectively bind with CD133 ＋ Huh-7 cells to regulate cell cycle and induce apoptosis.