目的：观察抗人类多药耐药相关蛋白3(multidrug resistance protein 3, MRP3)的单链抗体与可溶性坏死因子相关凋亡诱导配体（soluble TNF-related apoptosis inducing ligand, sTRAIL)的融合蛋白antiMRP3(scFv)-sTRAIL对多形性成胶质细胞瘤（glioblastoma multiforme，GBM）U251细胞凋亡的影响，并探讨其机制。方法：MTT法检测不同浓度antiMRP3(scFv)-sTRAIL（3.9063～250 nmol/L）作用后U251细胞的存活率。给予亲代antiMRP3(scFv)或TRAIL活性中和抗体mAb 2E5预孵育，应用IC50剂量的antiMRP3(scFv)-sTRAIL作用U251细胞，流式细胞仪检测U251细胞的凋亡率，Western blotting检测U251细胞中caspase-3的激活及其关键底物DNA裂解因子（DNA fragmentation factor，DFF）的降解。结果：随着antiMRP3(scFv)-sTRAIL浓度的增加，U251细胞的存活率逐渐降低\[（84.84±0.2）%～（19.93±1.8）%\]；IC50剂量（62.5 nmol/L）的antiMRP3(scFv)-sTRAIL单独或给予antiMRP3(scFv)、mAb 2E5预孵育后处理，致U251细胞的凋亡率分别为（58.0±1.3）%、（14.9±1.7）%和（17.4±3.0）%；antiMRP3(scFv)或mAb 2E5预孵育均可阻断U251细胞中antiMRP3(scFv)-sTRAIL诱导的caspase-3的激活及DFF的裂解。结论：AntiMRP3(scFv)-sTRAIL促进sTRAIL靶向识别并诱导GBM细胞凋亡，为开发靶向性抗肿瘤药物奠定了基础。

Objective: To observe the effects of antiMRP3(scFv)-sTRAIL on apoptosis in glioblastoma multiforme (GBM) U251 cells and to study its mechanism. Methods: Different concentration of antiMRP3(scFv)-sTRAIL (3.9063 nmol/L-250 nmol/L) was added into U251 cells, and then the viability was examined by MTT assay. IC50 dose of antiMRP3(scFv)-sTRAIL was added into U251 cells, in the presence or absence of parental antiMRP3(scFv) and the TRAIL-neutralizing mAb 2E5. The apoptotic percentage of U251 cells was examined by flow cytometry, and Western blotting was used to detect caspase-3 activation and DNA fragmentation factor (DFF) degradation in U251 cells. Results: When the concentration of antiMRP3(scFv)-sTRAIL increased (3.9063 nmol/L-250 nmol/L), the viability of U251 cells became lower synchronously (\[84.84±0.2\]%-\[19.93±1.8\]%); The apoptotic rates of U251 cells induced by IC50 dose (62.5 nmol/L) of antiMRP3(scFv)-sTRAIL or antiMRP3(scFv)-sTRAIL with parental antiMRP3(scFv) and mAb 2E5 were (58.0±1.3)%, (14.9±1.7)% and (17.4±3.0)%, respectively; antiMRP3(scFv) and the mAb 2E5 significantly inhibited the activation of caspase-3 and the degradation of DFF in U251 cells induced by antiMRP3(scFv)-sTRAIL. Conclusion: Targeted binding of antiMRP3(scFv)-sTRAIL to GBM increases the apoptosis induction activity of sTRAIL, which lays a foundation for further study on tumor-targeting drugs.

国家自然科学基金资助项目（No. 30571906）；国家新药创制科技重大专项课题资助项目（No. 2009ZX09102-234；No. 2009ZX09103-689）