黏蛋白1基因磁转染树突状细胞诱导细胞毒性T细胞抗膀胱肿瘤免疫效应的研究

Induction of specific CTL against bladder cancer by MUC1 gene-modified dendritic cells transfected with dextran coated magnetic iron oxide nanoparticles

目的:探讨黏蛋白1(mucins 1,MUC1)基因磁转染体外人树突状细胞(dendritic cell,DC)的可行性,观察其诱导的特异性抗MUC1膀胱癌CTL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基因的真核表达载体pEGFP-C1-MUC1,在钕-铁-硼稀土强磁块的磁场作用下转染DC,荧光显微镜下观察及流式细胞术检测转染效率,并用RT-PCR法检测转基因DC中MUC1基因的表达;再将转染MUC1基因的DC与自体T细胞共培养,并分别用乳酸脱氢酶释放法检测所致敏的细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL)对MUC1特异性抗膀胱癌(膀胱肿瘤BIU87细胞系)的杀伤活性,用透射电镜观察CTL诱导靶细胞凋亡情况;ELISA法测定MUC1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转染效率为10%左右,荧光显微镜下可观察到明显绿色荧光蛋白的表达,RT-PCR法可检测到MUC1条带,转染MUC1基因的DC与自体T细胞混合培养后能诱导出MUC1特异性的CTL,对BIU87细胞的杀伤实验表明T-DC-MUC1的杀伤活性显著高于对照组T-DC-pEGFP-C1和T-DC诱导的CTL(P均<0.05);在透射电镜下也可观察到部分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,明显高于未转染的DC(P<0.05)。结论:葡聚糖磁性纳米颗粒在固定磁场的作用下成功将MUC1基因转入DC,并可有效诱导出特异性抗MUC1膀胱癌的细胞毒性T细胞。

OBJECTIVE：To examine the ability of plasmid DNA encoding the human mucins 1（MUC1） to elicit antigen-specific CTL responses by gene transfer mediated by dextran coated magnetic iron oxide nanoparticles （DMN ） as gene carrier in vitro.METHODS：Dextran coated magnetic iron oxide nanoparticles（DMN ） modified with Poly-L-Lysine（PLL） as gene carrier transfected plasmid pEGFP-C1-MUC1 as the reporter gene into human dendritic cells（HDC ） in the magnetic field using Nd-Fe-B permanent magnet in vitro,and the rate of plasmid pEGFP-C1-MUC1 transfection into human dendritic cells were evaluated under fluorescence microscope,using RT-PCR and flow cytometer 24 h later.The transfected and nontransfected DC were then cocultured with autologous T cells,and with targeted bladder cancer cells（BIU87 ） seven days later.The cytotoxic activity or apoptosis of induced CTL to BIU87 was detected with LDH release assay or transmission electron microscope respectively.The IFN -γsecretion of the CTL was measured by ELISA assay.RESULTS：DMN acted as a vector in the magnetic field to transfect reporter gene pEGFP-C1-MUC1 into HDC.GFP was successfully expressed 24 h after transfection,and the rate of plasmid pEGFP-C1-MUC1 transfection was 10%.MUC1 expression was also detected as mRNA level in pEGFP-C1-MUC1 transfected DC.The transfected DC （DC-MUC1 ） successfully induced CTL with autologous T cells cocultured for seven days.The cytotoxic activity of induced CTL to BIU87 by T-DC-MUC1 was obviously higher than that by T-DC-pEGFP-C1 or T-DC.Transmission electron microscope showed apoptosis in the earlier period of CTL to BIU87 induction,for instance,disappearance of the nucleus、chromatin enriched around nuclear membrane,etc.There was significant difference in the ability of IFN -γsecretion between the transfected and nontransfected DC groups.CONCLUSION：Super-paramagnetic dextran coated magnetic iron oxide nanoparticles（DMN ） as a vector could transfect plasmid pEGFP-C1-MUC1 into HDC MUC1 protein could enhance the ability of DC to stimulate autologous T cells proliferation and induce most potent cytotoxicity of CTL to bladder cancer cells（BIU87 ）.