^(131)I标记CD133单链抗体对人肝癌CD133^+ HepG2干细胞的抑制作用

Inhibitory effect of ^(131)I-labeled anti-CD133 ScFv on CD133^+ human hepatocellular carcinoma cells

目的:研究131I标记CD133单链抗体(single chain rariable fragment,ScFv)在体外对人肝癌CD133+HepG2干细胞的抑制作用。方法:免疫磁珠分选HepG2细胞,流式细胞术检测分选前后HepG2细胞的CD133表达率,克隆形成实验及体内成瘤实验验证CD133+HepG2细胞的"干性"。氯胺T法131I标记CD133 ScFv并测定标记率、比活度、放射性浓度。将分选出的CD133+HepG2细胞分为131I-CD133抗体治疗组、131I治疗组、CD133抗体治疗组和131I+CD133抗体治疗组,MTT法检测各组中对CD133+HepG2细胞生长抑制的最适剂量和不同药物在12、48、72 h三个时间点对CD133+HepG2干细胞的生长抑制作用,流式细胞术检测各组细胞周期的变化。结果:分选的HepG2细胞的CD133表达率显著高于未分选细胞[(97.71±1.13)%vs(1.52±0.78)%,t=1.13、P=0.000]。CD133+HepG2细胞相对于CD133-HepG2细胞具有更强的体外成球、克隆形成能力[(45.03±1.35)%vs(7.4±0.54)%;t=3.92,P=0.000]和体内成瘤能力。131I-CD133 ScFv的标记率为88.92%,放射化学纯度为98.63%。当131I为3.7 MBq/100μl、CD133抗体为1μg/100μl时,对CD133+HepG2细胞的抑制率最高,达(89.58±0.74)%;在此剂量下131I-CD133 ScFv治疗组对CD133+HepG2细胞生长抑制率显著高于其余各实验组,且呈时间依赖性。131I-CD133 ScFv治疗组G0/G1期细胞比例为(27.50±1.12)%,较其余各组均明显减少(P<0.05)。结论:成功制备的131I-CD133 ScFv在体外能有效抑制人肝癌CD133+HepG2干细胞的生长。

Objective：To study the inhibitory effect of the anti-CD133 single chain variable fragment （ScFv） labeled with 131I on CD133＋cancer stem cells （CSCs） sorted form human hepatocellular liver carcinoma HepG2 cells in vitro . Methods： CD133＋ and CD133- CSCs were isolated from HepG2 cells through magnetic-activated cell sorting （MACS）. CD133 expression in both sorted and unsorted cells was analyzed by flow cytometry （FCM）. The property of CD133＋ CSCs was validated by sphere-forming assay and colony formation assay in vitro and tumor formation assay in nude BALB/c mice in vivo . The monoclonal antibody CD133 was labeled with 131I using the chloramines T method and the labeling rate, specific activity and radioactivity were evaluated. CD133＋ CSCs were treated with 131I, CD133 ScFv, 131I-CD133 ScFv, and 131I＋CD133 ScFv. At 12, 24 and 48 hours after treatment, cell proliferation and cell cycle progression were assessed by MTT assay and FCM respectively. Results： CD133 was detected in （97.71±1.13）% of the sorted CD133＋ HepG2 cells but in only （1.52±0.78）% of unsorted HepG2 cells （ P =0.0001）. As compared with CD133- HepG2 cells, CD133＋ HepG2 cells showed a higher tumor sphere formation ability （[45.03±1.35]% vs [7.4±054]%, P 〈0.001）. The 131I labeling rate of CD133 ScFv was 88.92%, and the radiochemical-purity was 98.63%. A maximal CD133＋ cell growth inhibition of （89.58±0.74）% was observed （ P 〈0.05） when 131I was used at 3.7 MBq/100 μl and CD133 ScFv was used at 1 μg/100 μl, significantly higher than other doses （ P 〈0.05）. The proportion of G0/G1 phase arrest in cells treated with 131I-CD133 ScFv was significantly reduced as compared with treatments （ P 〈0.05）. Conclusion： Radioisotope 131I labeled CD133 ScFv may effectively inhibit growth of CD133-positive human hepatocellular carcinoma cells in vitro . 