CAJ | 학술논문

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。
이중조선상관병독재체（recombinant adeno -associated virus，rAAV）휴대증강형록색형광단백（enhanced green fluorescent protein，EGFP）기인전염입대서저수，관찰기정위급표체정황，평개기작위저수병변기인치료재체적가행성。SD 대서경방광기층다점주사 rAAV2/1-EGFP，분별안전염복수1×109、1×1010、4×1010전염。분별재2주、4주、8주후，장대서심마취，경좌심실-승주동맥삽관4％다취갑철관주고정，만만박리출척수，장척수방입함30％자당적 PBS 중과야（4℃），연후항온빙동절편궤내련속절편，후도12μm，면역형광염색（GFP 항체，FITC 표기산양항토 IgG）후，재형광현미경하관찰 EGFP 표체정황。형광현미경하현시，4주시，전염복수위4×1010적대서저수신경원표체강형광，가견증강형록색형광단백미산표체。rAAV2/1재체가장 EGFP 기인유효지전염입대서저수，rAAV2/1재체시일충이상적기인치료재체，위rAAV2/1작위재체전염저수치료신경원성질병제공이론의거。
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.