联合输注Flt3-L和GM-CSF表达质粒体内诱导小鼠DC的研究

Identification of mouse dentritic cells expanded in vivo by combined over-expression of Flt3-L with GM-CSF

目的:探讨联合输注Flt3-L和GM-CSF真核表达质粒体内诱导小鼠DC的方法并检测其抗原提呈功能。方法:采用尾静脉注射法分别输注Flt3-L及GM-CSF质粒,15d后收获小鼠脾脏;采用流式细胞术检测小鼠脾细胞CD11c、CD11b、B220、CD8α和NK1.1等表面标志以鉴定DC的比例及亚型;将体内诱导DC与HBsAg共孵育,然后刺激HBsAg预免疫小鼠的脾细胞并检测其分泌IFN-的水平。结果:联合输注Flt3-L和GM-CSF质粒小鼠的脾细胞达到4×108个细胞/脾脏、其中CD11c+细胞占20%以上,CD11c+细胞中CD11b+、CD11b-、B220+、CD8+、NK1.1+细胞的比例分别达17%、10%、26%、16%、7%左右;体内诱导DC负载HB-sAg后刺激HBsAg特异性淋巴细胞分泌IFN-γ的水平明显高于HBsAg单独刺激组。结论:采用小鼠尾静脉注射技术输注Flt3-L及GM-CSF质粒能够诱导CD11c+ DC的产生并包含多种亚型,体内诱导DC具备有效的抗原提呈功能。

AIM：To establish a method to expand dentritic cells （DC） in vivo by combined injection of Flt3-L-expressing plasmid with GM-CSF-expressing plasmid and to analyze their function in antigen presenting. METHODS： Flt3-L and GM-CSF expressing DNAs were injected into the tail veins of BALB/c mice on day 0 and day 10 respectively. The spleens were harvested on day 15 and the splenocytes were prepared to analyze the percentage of CD11c, CD11b, B220, CD8, and NK1.1 positive cells by FACS. The splenocytes from HBsAg-vaccinated mice were stimulated in vitro with DC pre-incubated with HBsAg and then IFN-γ in supernatant after stimulation was detected by ELISA. RESULTS： Combined injection of Flt3-L-expressing plasmid with GM-CSF-expressing plasmid resulted in dramatic splenomegaly in mice. Averagely 4×108 splenocytes were harvested from the expanded spleen of each mouse, more than 20% of which were CD11c＋ DC. Among CD11c＋ splenocytes, 17% of CD11b＋ cells, 10% of CD11b-cells, 26% of B220＋ cells, 16% of CD8＋ cells and 7% of NK1.1＋ cells were detected respectively. When loaded with HBsAg, Flt3-L/GM-GSF DC could stimulate HBsAg-primed splenocytes to secrete much higher IFN-γ than DC or HBsAg alone. CONCLUSION： Combined intravenous injection of Flt3-L-expressing plasmid with GM-CSF-expressing plasmid could in vivo expand dentritic cells which seemed to have many subsets and were able to present antigens to antigen special T cells in vitro.