【摘要】 利用减毒沙门氏菌为载体将4-1BBL体内转染抗原提呈细胞,观察体内4-1BBL基因转染对大鼠免疫功能的影响。构建含pIRES2-EGFP-4-1BBL质粒的减毒沙门氏菌(疫苗菌),通过体外感染HepG2细胞观察疫苗菌的侵袭功能和携带外源基因能力,验证疫苗菌作为4-1BBL基因转染载体的作用。疫苗菌2周内连续灌服大鼠3次,末次灌服后14 d心脏取血处死,RT-PCR和荧光显微镜检测报告绿色荧光蛋白(GFP)在脾脏细胞中的转录表达,流式细胞仪检测外周血和脾脏中细胞表型变化。疫苗菌在体外能成功的将外源基因携带至HepG2细胞进行表达,灌服疫苗菌的大鼠外周血和脾脏细胞4-1BBL和4-1BB表达明显升高,外周血中CD3+、CD3+CD8+和CD3+CD25+,脾脏细胞中CD3+CD8+、CD3+CD25+也有明显的增高。表明含4-1BBL基因的减毒沙门氏疫苗菌能实现体内基因转染,4-1BBL的高表达能增强机体的细胞免疫功能。更多还原

【Abstract】 The 4-1BB ligand(4-1BBL) expressed on antigen-presenting cells(APC) interacts with 4-1BB on activated T cells(especially CD8+ cells) and co-stimulates the latter to secrete cytokines and to proliferate.To date,there are only limited data on APC transfection in vivo.To further explore the 4-1BBL gene transfer in vivo and to understand the effect of 4-1BBL gene transfer in immunoreaction.A recombinant vaccine bacteria containing 4-1BBL were generated and the invasion of these vaccines were detected in vitro by incubation with HepG2 cells.This vaccines were then fed on rat with 3 times in 2 weeks,and 14 days after the end of irrigation,the level of phenotype change in peripheral blood cells and spleen cells was determined by flow cytometry.It was demonstrated that this recombinant vaccine containing 4-1BBL in vitro could successfully carry foreign gene and resulted in expression of rat 4-1BBL on HepG2 cells.Report gene of the green fluorescent protein(GFP) in splenocytes were detected by RT-PCR and fluorescent microscopy.There were significantly increased expression of 4-1BBL and 4-1BB in peripheral blood cells(PBC)and splenocytes.The up-regulation of CD3+,CD3+CD8+,and CD3+CD25+ in PBC and CD3+CD8+,CD3+CD25+on spleen cells were also detected.Thus,this vaccine can achieve APC with 4-1BBL gene transfected in vivo,which is a promising adjuvant to enhance immune function.更多还原