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目的:观察HA纳米颗粒载体介导转染hGM-CSF基因的HepG2细胞疫苗体外抗肿瘤效应,为hGM-CSF基因修饰的HepG2细胞疫苗的临床应用提供依据。方法:HA纳米颗粒载体介导hGM-CSF基因转染HepG2细胞制备转GMCSF基因的HepG2细胞疫苗。密度梯度离心法分离人PBMC,体外诱导人PBMC。WST-1法测定PBMC的增殖活性及对HepG2细胞的杀伤效应,流式细胞术分析CD4+和CD8+的阳性表达率,ELISA法测定INF-γ的分泌。结果:WST-1结果显示,转基因HepG2组疫苗能诱导PBMC增殖,其增殖率优于野生型疫苗(p<0.05);其诱导的PBMC对HepG2的杀伤率高于各野生型疫苗组和各空白对照组(p<0.05)。FCM结果显示,转基因HepG2疫苗组PBMC中CD4+和CD8+阳性表达率均高于各野生型疫苗组和各空白对照组(p<0.05)。ELISA结果显示,转基因组PBMC培养上清中IFN-γ含量为1989.76±254.21pg/ml,高于各野生型疫苗组和各空白对照组(p<0.05)。结论:HA纳米颗粒载体介导转染hGM-CSF基因能增加HepG2细胞疫苗的免疫原性,转hGM-CSF基因HepG2细胞疫苗可有效诱导PBMC增殖、分化,增加INF-γ的分泌,提高其对HepG2细胞的杀伤作用。
OBJECTIVE: To observe the antitumor effect of HA nanoparticle vector-mediated HepG2 cell vaccine transfected with hGM-CSF gene in vitro and to provide evidence for the clinical application of hGM-CSF gene modified HepG2 cell vaccine. Methods: HepG2 cells were transfected into HepG2 cells by HA nanoparticle vector mediated gene transfer of HepG2 cells. Human PBMCs were isolated by density gradient centrifugation and induced in vitro. The proliferative activity of PBMC and its killing effect on HepG2 cells were determined by WST-1 assay. The positive rates of CD4 + and CD8 + were analyzed by flow cytometry. The secretion of INF-γ was measured by ELISA. Results: The results of WST-1 showed that the HepG2 vaccine could induce the proliferation of PBMCs, and its proliferation rate was higher than that of wild-type vaccine (p <0.05). The killing rate of HepG2 induced by PBMCs was higher than that of wild-type vaccines and blank Control group (p <0.05). The results of FCM showed that the positive rate of CD4 + and CD8 + in PBMC of transgenic HepG2 vaccine group was higher than that of wild-type vaccine group and each blank control group (p <0.05). The results of ELISA showed that the IFN-γ content in the supernatant of the PBMC of the transgenic group was 1989.76 ± 254.21 pg / ml, higher than that of each wild-type vaccine group and each blank control group (p <0.05). CONCLUSION: HA nanoparticle vector-mediated transfection of hGM-CSF gene can increase the immunogenicity of HepG2 cell vaccine. The HepG2 cell vaccine can effectively induce the proliferation and differentiation of PBMC and increase the secretion of INF-γ HepG2 cell killing effect.