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/100 μl、CD133抗体为1 μg/100 μl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G0/G1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 G0/G1 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. 

国家自然科学基金资助项目(No.30370422，No.81171365)