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目的构建HIV-1C亚型gp120负载人树突状细胞(dentriti ccell,DC)疫苗,并对其体外功能进行初步检测。方法利用Amaxa细胞核转染技术将pcDNA3.1-gp120质粒转染至人成熟DC,以Western blot检测gp120的表达。通过流式细胞仪检测DC表面共刺激分子的变化、混合淋巴细胞反应、CD8+T细胞表面活化分子CD25的表达及其分泌IFN-γ的变化。结果通过Western blot检测,gp120在DC中得到了正确表达。经流式细胞仪检测,DC表面分子CD80表达率由刺激前的33.34%上升至43.20%,CD86表达率由刺激前的60.08%上升至90.34%;负载gp120DC刺激淋巴细胞增殖率为86.72%;CD8+T细胞表面分子CD25表达率由刺激前的5.27%上升至74.21%,IFN-γ的表达率达37%。结论负载了HIV-1gp120的人树突状细胞能够显著刺激淋巴细胞的增殖、增强CD8+T细胞表面活化分子CD25表达以及促进CD8+T细胞分泌IFN-γ,为下一步DC治疗性疫苗的体内研究奠定基础。
Objective To construct a HIV-1 subtype gp120-loaded human dendritic cell (DC) vaccine and to perform preliminary in vitro functional tests. Methods The pcDNA3.1-gp120 plasmid was transfected into human mature DC by Amaxa nuclear transfection and the expression of gp120 was detected by Western blot. Flow cytometry was used to detect the changes of DC surface costimulatory molecules, mixed lymphocyte reaction, CD8 + T cell surface activation molecule CD25 expression and secretion of IFN-γ. Results Western blot showed that gp120 was correctly expressed in DC. Flow cytometry showed that the expression rate of CD80 on DCs increased from 33.34% before stimulation to 43.20%, while the expression of CD86 increased from 60.08% to 90.34% before stimulation. The proliferation rate of lymphocytes stimulated by gp120DC was 86.72% + T cell surface molecules CD25 expression increased from 5.27% before stimulation to 74.21%, IFN-γ expression rate of 37%. Conclusion Human dendritic cells loaded with HIV-1gp120 can significantly stimulate the proliferation of lymphocytes, enhance the expression of CD25 on CD8 + T cells, and promote the secretion of IFN-γ by CD8 + T cells. Research laid the foundation.