rAAV2/1载体介导EGFP对大鼠骶髓转染的实验研究  

The Experimental Research of rAAV2/1 Vector Mediated Transfection of Enhanced Green Fluorescent Protein on the Sacral Cord

作　　者：胡映秋[1] 徐美玲[2] 

机构地区：[1]南昌大学第四附属医院,江西南昌330000 [2]南昌大学第二附属医院,江西南昌330000

出　　处：《生物医学工程研究》2013年第4期239-242,共4页Journal Of Biomedical Engineering Research

摘　　要：以重组腺相关病毒载体(recombinant adeno-associated virus,rAAV)携带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因转染入大鼠骶髓,观察其定位及表达情况,评价其作为骶髓病变基因治疗载体的可行性。SD大鼠经膀胱肌层多点注射rAAV2/1-EGFP,分别按转染复数1×109、1×1010、4×1010转染。分别在2周、4周、8周后,将大鼠深麻醉,经左心室—升主动脉插管4%多聚甲醛灌注固定,慢慢剥离出脊髓,将脊髓放入含30%蔗糖的PBS中过夜(4℃),然后恒温冰冻切片机内连续切片,厚度12μm,免疫荧光染色(GFP抗体,FITC标记山羊抗兔IgG)后,在荧光显微镜下观察EGFP表达情况。荧光显微镜下显示,4周时,转染复数为4×1010的大鼠骶髓神经元表达强荧光,可见增强型绿色荧光蛋白弥散表达。rAAV2/1载体可将EGFP基因有效地转染入大鼠骶髓,rAAV2/1载体是一种理想的基因治疗载体,为rAAV2/1作为载体转染骶髓治疗神经源性疾病提供理论依据。To recombine adeno -associated virus vector (recombinant adeno -associated virus,rAAV)carrying enhanced green fluorescent protein (enhanced green fluorescent protein,EGFP)gene into the rat sacral cord,observe the location and expression and evaluate it as the feasibility of gene therapy vector of sacral cord lesion.SD rats were injected rAAV2 /1 -EGFP in bladder muscle by more,according to different multiplicity of infection (1 ×109、1 ×10 10、4 ×10 10 ).The rats were deeply anesthetized after two weeks, four weeks,eight weeks and fixed by 4% paraformaldehyde with the left ventricle -aorta cannulation,the spinal cord was slowly stripped and put into the PBS containing 30% sucrose in the night (4 ℃)and serial sectioned within the frozen section machine at constant temperature,the thickness was 12 μm and stained with immunofluorescence (GFP antibody,FITC labeled goat anti -rabbit IgG),the EGFP fluorescence expression was observed under the microscope.Fluorescence microscopy revealed that four weeks later,the multiplicity of infection of 4 ×1010 had strong fluorescence of sacral cord neurons,showing the enhanced expression of green fluorescent protein diffusion.EGFP gene can be effectively transfected into rat sacral cord by rAAV2 /1 vector ,rAAV2 /1 vector is an ideal gene ther-apy vectors.For rAAV2 /1 vector is transfected as the treatment of sacral nerve -born diseases and provide a theoretical basis.

关 键 词：重组腺相关病毒 增强型绿色荧光蛋白 骶髓 转染 基因治疗 

分 类 号：R318[医药卫生—生物医学工程]