脐带间充质干细胞运载scFvCD20：sTRAIL融合蛋白对B-淋巴瘤细胞的生长抑制作用

Human umbilical cord-drived mesenchymal stem cells as vehicles of CD20 specific-TRAIL fusion protein against non-Hodgkin's lymphoma

目的:探讨脐带间充质干细胞运载scFvCD20:s TRAIL融合蛋白的新型双重靶向系统对CD20+BJAB细胞的生长抑制作用。方法:采用传统分子生物学技术构建p LenR.sc FvCD20:s TRAIL、p LenR.ISZ-s TRAIL、p LenR.sc Fv CD20及p LenR.cop GFP四种慢病毒表达载体,利用四质粒慢病毒包装系统于293T细胞中包装慢病毒颗粒,并感染人脐带组织来源的MSCs(HUMSCs),使其稳定表达融合蛋白。于体外采用CCK8细胞增殖抑制实验检测sc Fv CD20:s TRAIL融合蛋白对CD20阳性BJAB细胞和Raji细胞、CD20阴性Jurkat细胞以及正常人外周血单个核细胞(PBMCs)的生长抑制作用。建立NOD/SCID鼠BJAB细胞皮下移植瘤模型,将MSC.sc Fv CD20:s TRAIL经尾静脉注射入小鼠体内,每3 d测量瘤体积,根据肿瘤体积计算抑瘤率。结果:成功构建了慢病毒表达载体p LenR.sc Fv CD20:s TRAIL、p LenR.ISZ-s TRAIL、p LenR.sc Fv CD20及p LenR.cop GFP,且经慢病毒感染可在HUMSCs中稳定表达。体外实验显示,sc Fv CD20:s TRAIL融合蛋白可不同程度地提高对CD20阳性BJAB和Raji细胞的生长抑制作用,而对CD20阴性Jurkat细胞的生长抑制作用降低;而且不影响PBMCs的生长。体内实验表明,MSC.sc Fv CD20:s TRAIL可显著抑制BJAB淋巴瘤的生长,初始治疗后第24天,抑瘤率达65.2%,与MSC.ISZ:s TRAIL治疗组比较(抑瘤率为52.7%),具统计学差异(P<0.05)。结论:建立了HUMSCs运载sc Fv CD20:s TRAIL融合蛋白的双重靶向治疗系统,HUMSCs可向BJAB淋巴瘤部位归巢并表达分泌sc Fv CD20:s TRAIL融合蛋白,后者在局部经sc Fv CD20的二次导向发挥CD20特异性抑瘤作用。为MSCs作为肿瘤靶向治疗载体在临床中的应用提供了理论依据。

Objective： To study the therapeutic effect of a novel double-target system,in which human umbilical cord-derived MSCs were used as vehicles to deliver fusion protein scFvCD20： sTRAIL to non-Hodgkin＇s lymphoma. Methods： The traditional methods in molecular biology were used to construct lentivirus expression vectors p LenR. scFvCD20： sTRAIL and contrast vectors. Human umbilical cord-derived MSCs（ HUMSCs） were labeled with the cop GFP by transducing with pseudo viral particles which had been packaged in 293 T cells with four plasmid-lentivirus packaging system. Fusion protein scFvCD20： sTRAIL were secreted from MSC. scFvCD20： s TRAIL after that HUMSCs were infected by pseudo viral particles. CCK8 assay was applied to detect the antigen-restricted cell death induced by sc Fv CD20： s TRAIL in CD20-positive BJAB and Raji cells as well as CD20-negtive Jurkat cells and human normal peripheral blood mononuclear cells（ PBMCs）. To evaluate the therapeutic effect of MSC. scFvCD20： s TRAIL in vivo,genetically modified HUMSCs were intravenously injected into tumor-bearing mice with BJAB cells. The volume of tumor was measured every three days,and the inhibition ratio of tumor was calculated according to tumor volume. Results： Lentivirus expression vectors p LenR. scFvCD20： sTRAIL,p LenR. ISZ： sTRAIL,p LenR. scFvCD20 and p LenR. Cop GFP were successfully constructed and these constructs could be expressed stably in HUMSCs by lentivirus transduction. scFvCD20： s TRAIL fusion protein produced a potent inhibition of cell proliferation in CD20-positive BJAB cells,moderate inhibition of the growth of Raji cells,and weak inhibition in CD20-negtive Jurkat cells when compared with ISZ-s TRAIL treatment,and it had no effect on normal human peripheral blood mononuclear cells（ PBMCs）. The MSC. scFvCD20： sTRAIL treatment significantly inhibited the tumor growth when compared with those treated with MSC. ISZ-s TRAIL. Conclusion： A double-target therapeutic system is well established,in which HUMSCs migrated to tumor site,secreted a novel fusion protein scFvCD20： s TRAIL,and thus locally concentrated scFvCD20： sTRAIL extended antigen-restricted antitumor activity. The engineered HUMSCs secreting sc Fv CD20： sTRAIL showed potent effect on inhibiting tumor growth in BJAB lymphoma malignancy,which may play an essential role in the clinical research.