磁转染MUC1/Y基因入树突状细胞抗膀胱肿瘤的免疫效应研究

The induction of specific CTL against bladder cancer by MUC1/Y gene-modified dendritic cells which transfected with dextran coated magnetic iron oxide nanoparticles in the magnetic field

目的:研究体外磁转染人MUC1/Y基因至人树突状细胞(DC)的可行性,以及在体外诱导特异性抗MUC1/Y膀胱癌的免疫效应。方法:以葡聚糖磁性纳米颗粒(DMN)作为载体,在多聚赖氨酸(PLL)的辅助下,通过静电作用结合MUC1/Y基因的真核表达载体pEGFP-C1-MUC1/Y,在铜-硼-锡磁块的固定磁场作用下转染至人DC中,荧光显微镜下以及流式细胞仪观察其转染效率;再将这种转基因DC与自体T细胞共培养,观察其致敏的细胞毒性T淋巴细胞(CTL)对MUC1/Y特异性抗膀胱癌(膀胱肿瘤BIU87细胞系)的杀伤活性,即分别用LDH释放法检测CTL杀伤活性和透射电镜观察CTL诱导靶细胞凋亡情况;ELISA法测定基因修饰后的DC刺激自体T细胞分泌IFN-γ的能力。结果:pEGFP-C1/-MUC1/Y转染效率为15%左右,荧光显微镜下可观察到明显绿色荧光蛋白的表达;与自体T细胞混合培养后能诱导出显著的MUC1/Y特异性的CTL,对BIU87细胞的杀伤实验表明T-DC-MUC1的杀伤活性约为52%,显著高于对照组T-DC诱导的CTL;在透射电镜下也可以清楚的观察到部分BIU87膀胱肿瘤细胞出现了细胞核核仁消失,染色质浓集于核膜周围等早期凋亡表现;基因修饰后的DC能刺激自体T细胞分泌高水平的IFN-γ,与未转染的DC相比差异有统计学意义(P<0.05)。结论:葡聚糖磁性纳米颗粒(DMN)在固定磁场的作用下成功将MUC1/Y基因转入DC,并可有效诱导出特异性的抗MUC1/Y膀胱癌的免疫效应。

Objective：To examine the ability of plasmid DNA encoding the human MUC1/Y to elicit antigenspecific CTL responses by gene transfer mediated by dextran coated magnetic iron oxide nanoparticles （DMN） as gene carrier in the magnetic field in vitro. Methods： The dextran coated magnetic iron oxide nanopartieles （DMN） modified with Poly L-Lysine （PI.L） as gene carrier transfect plasmld pEGFP-C1 MUC1/Y as the reporter gene in to human dendritic cells （HDC） in the magnetic field using Nd-Fe-B permanent magnet in vitro, and the rate of transfection of plasmid pEGFP C1-MUC1/Y into human dendritic cells are evaluated under fluorescence microscope and flow cytometer 24 hours later. After transfection . the transfected and nontransfected DC are cocultured with autogenetic T cells respectively . and seven days later with targeted bladder cancer cells （BIU87）, The cytotoxic activity or apoptosis of induced CTI. to BIU87 is detected with LDH release assay or transmission electron microscope respectively. The IFN-γ secretion of the CTL are detected by EI.ISA assays. Results：DMN acts as a vector in the magnetic field to transfect reporter gene pEGFP-C1 MUC1/Y into HDC and GFP is successfully expressed, 24halter transfection . and the rate of transfectionof plasmid pEGFP-C1 MUC1/Yis 15%. The transfected DC （DC- MUC1/Y ） can successfully induced CTL with autogenetic T cells cocuhured seven days later. The cytotoxic activity of induced CTL to BIU87 by T-DC-MUC1/Y is obviously higher than that by T DC, Trans mission electron microscope map show apoptosis in the earlier period of induced CTL to BIU87, for instance, ento- blast disappearance.chromatin enriched around nuclear membrane, et al. There is significant difference in the ability of IFN-γ secretion between the transfected and nontransfected DC groups. Conclusions： Superparamagnetic dextran coated magnetic iron oxide nanoparticles （DMN） as a vector can transfect plasmid pEGFP-C1 MUC1/Y into HDC in the magnetic field successfully, and MUC1/Y protein can enhance the ability of DC to stimulate autogenetic T cells proliferation and induce most potent cytotoxicity of CTL to bladder cancer cells（BIU87）.