论文部分内容阅读
目的:研究热休克蛋白65(HSP65)-前列腺特异性抗原融合蛋白在体外对人树突状细胞的活化作用以及在小鼠体内抑制PSA阳性肿瘤细胞生长的作用。方法:利用Westernblot方法对融合蛋白HSP65-PSA的特异性进行鉴定;利用共聚焦显微镜、流式细胞术(FCM)检测和斑点杂交的方法对PcDNA3-GFP-PSA稳定转染的B16细胞进行鉴定;用HSP65-PSA融合蛋白刺激来自于人外周血的未成熟树突状细胞,观察融合蛋白对树突状细胞的体外活化作用;用PcDNA3-GFP-PSA稳定转染的B16细胞种植C57BL/6小鼠,继而用HSP65-PSA融合蛋白来免疫小鼠以观察融合蛋白对PSA阳性B16肿瘤细胞生长的体内抑制作用。结果:Westernblot检测显示融合蛋白HSP65-PSA具有PSA蛋白特异性。共聚焦显微镜、FCM检测和斑点杂交结果均显示PcDNA3-GFP-PSA转染的B16细胞转染情况良好。体外细胞实验显示,将HSP65-PSA融合蛋白作用于人外周血树突状细胞后可以明显促进树突状细胞表面分子CD86的表达,其上调率为52.93%。动物体内实验显示,HSP65-PSA融合蛋白可以明显抑制PSA阳性B16肿瘤细胞在小鼠体内的生长。10μg蛋白免疫组的肿瘤体积(4.91±5.21)与对照组(10.56±6.10)相比差异有统计学意义(P<0.05)。结论:HSP65-PSA融合蛋白可以在体外活化树突状细胞,在小鼠体内抑制PSA阳性B16肿瘤细胞的生长。HSP65-PSA融合蛋白可能具有应用于PSA阳性肿瘤的免疫治疗作用。
AIM: To investigate the activation of human dendritic cells (HSPs) and prostate-specific antigen (GFP) fusion proteins in vitro and the inhibition of the growth of PSA-positive tumor cells in mice. Methods: Western blotting was used to identify the specificity of fusion protein HSP65-PSA. B16 cells stably transfected with PcDNA3-GFP-PSA were identified by confocal microscopy, flow cytometry (FCM) and dot blotting. The immature dendritic cells from human peripheral blood were stimulated with HSP65-PSA fusion protein to observe the activation of dendritic cells in vitro. B16 cells stably transfected with PcDNA3-GFP-PSA were planted with C57BL / 6 Mice were then challenged with HSP65-PSA fusion protein to observe the in vivo inhibition of the fusion protein on PSA-positive B16 tumor cell growth. Results: Western blotting showed that the fusion protein HSP65-PSA possessed PSA specificity. Confocal microscopy, FCM and dot blot hybridization results showed that transfection of PcDNA3-GFP-PSA-transfected B16 cells was good. In vitro experiments showed that the HSP65-PSA fusion protein could significantly promote the expression of CD86 on dendritic cells, which was up-regulated by 52.93%. In vivo experiments in animals show that the HSP65-PSA fusion protein can significantly inhibit the growth of PSA-positive B16 tumor cells in mice. The tumor volume (4.91 ± 5.21) in 10μg protein immunized group was significantly different from that in control group (10.56 ± 6.10) (P <0.05). Conclusion: HSP65-PSA fusion protein can activate dendritic cells in vitro and inhibit the growth of PSA-positive B16 tumor cells in mice. HSP65-PSA fusion proteins may have immunotherapy effects for PSA-positive tumors.