中文摘要：

目的探讨基因枪颗粒轰击转染人树突细胞（DC）的方法,以及基因枪转染基因疫苗质粒IgHV1-GM-CSF/pcDNA3.0在DC中的表达。方法利用Ficoll密度梯度法分离人外周血单个核细胞（PBMC）,贴壁方法获取单核细胞作为DC前体,在重组GM-CSF和IL-4的支持下诱导培养为用于转染的未成熟DC（i mDC）;制备人免疫球蛋白重链基因可变区家族的框架区基因疫苗质粒和增强绿色荧光蛋白质粒（pEGFP）。设定不同的转染条件,采用基因枪方法将pEGFP质粒转染入DC,荧光显微镜下分别计数绿色荧光阳性细胞数和细胞总数,计算转染率;台盼蓝染色计数活细胞,计算细胞存活率;比较基因枪、脂质体及电转染3种方法的转染效率。以基因枪方法在最佳条件下将IgHV1-GM-CSF/pcDNA3.0及pEGFP质粒共转染DC,提取细胞RNA,RT-PCR方法检测IgHV1-GM-CSF的表达,ELISA方法检测GM-CSF的表达。结果选择基因枪转染的优化条件为：转染压力120psi、聚乙烯吡咯烷酮（PVP）浓度0.02mg/ml、微载体负载量（MLQ）0.5mg金/子弹、DNA负载率（DLR）1.5μg质粒DNA/子弹。基因枪转染的效率为10.5%±2.4%（n=3）,电转染方法为45.2%±5.6%（n=3）,而脂质体方法只有个别DC表达荧光蛋白,三者比较差异有统计学意义（P〈0.01）。基因枪方法转染的DC能够保持其特殊细胞形态,细胞表面标志在转染中无明显变化,而电转染后细胞大量死亡。应用基因枪转染方法,基因疫苗质粒IgHV1-GM-CSF/pcDNA3.0在DC中得到瞬时表达。结论基因枪颗粒轰击可使基因疫苗在DC中成功表达,是临床细胞免疫治疗中DC转染的一种可行选择。

英文摘要：

Objective To explore the application of particle-gun technique in transfecting dendritic cells （DCs）, and investigate the expression of DNA vaccine particle IgHV1-GM-CSF in DCs induced by particle-gun. Methods Monocytes were isolated from donor peripheral blood collected by Ficoll density gradient method and adherent culture, and then differentiated into immature DCs （imDCs） by rhGM-CSF and rhIL-4 induction. The plasmids for transfection were IgHV1-GM-CSF/pcDNA3.0 and pEGFP, which were prepared with endofree plasmid purification kit. The DCs were transfected with plasmid pEGFP by particle-gun at different transfection conditions, the green fluorescence positive cell and the total cell numbers were counted respectively under fluorescence microscope, and the transfection efficiency was calculated; the viable cell count was performed after trypan blue staining; the transfection efficiencies of particle-gun, liposome-mediated transfection and electroporation method were compared. The plasmids IgHV1-GMCSF/pcDNA3. 0 and pEGFP were cotransfected into imDCs by particle-gun under the optimum conditions, and then DNA was extracted, the expression of IgHV1-GM-CSF was determined by RT-PCR, and of GM-CSF by ELISA. Results The optimum conditions of particle-gun transfection in imDCs were： gas pressure at 120psi, PVP concentration in 0. 02mg/ml, microcarrier loading in 0. 5mg gold/shot, and DNA loading ratio in 1.5μg plasmid/mg gold. The transfection efficiencies of particle-gun and electroporation were 10. 5%±2. 4% （n=3） and 45.2%±5. 6% （n=3） respectively, while that of liposome-mediated transfection was very low, and significant difference existed among the three methods （P〈 0. 01）. Furthermore, imDCs transfected with particle-gun could retain the special morphology, and its surface markers had no markedly change, while most of the cells died after transfection by electroporation method. Transient expression of gene vaccine IgHV1-GM-CSF/ pcDNA3. 0 in imDCs was observed after particle-gun transfecti