CAJ | 학술논문

目的探索高效、安全的自分泌运动因子(autocrine motility factor,AMF)基因转染方法 ,为携带AMF基因的成肌细胞移植提供实验依据. 方法取SD大鼠胸肌,用组织块培养法原代培养成肌细胞,纯化、鉴定、扩增成肌细胞;构建携带AMF及增强型绿色荧光蛋白(enhancedgreen fluorescent protein,EGFP)基因的猫免疫缺陷病毒(feline immuneddieiency vires,FIV)慢病毒载体;后者转染至成肌细胞;用荧光显微镜、激光共聚焦显微镜检测EGFP以确定转染的阳性率;应用免疫组化方法检测AMF的表达. 结果经过2周的原代培养及纯化,可获得纯度为98%的成肌细胞,在转染复数(multiplieity ofinfection,MOI)为100时,可获得90.4%(P<0.01)的转染阳性率,而转染后的AMF基因能正常表达. 结论组织块培养法适合成肌细胞的原代培养;FIV载体能以高转染率将AMF基因转至大鼠成肌细胞,并获得高效的表达.该方法为一种较理想的AMF基因转染模式.
목적 탐색고효、안전적자분비운동인자(autocrine motility factor,AMF)기인전염방법 ,위휴대AMF기인적성기세포이식제공실험의거. 방법 취SD대서흉기,용조직괴배양법원대배양성기세포,순화、감정、확증성기세포;구건휴대AMF급증강형록색형광단백(enhancedgreen fluorescent protein,EGFP)기인적묘면역결함병독(feline immuneddieiency vires,FIV)만병독재체;후자전염지성기세포;용형광현미경、격광공취초현미경검측EGFP이학정전염적양성솔;응용면역조화방법 검측AMF적표체. 결과 경과2주적원대배양급순화,가획득순도위98%적성기세포,재전염복수(multiplieity ofinfection,MOI)위100시,가획득90.4%(P<0.01)적전염양성솔,이전염후적AMF기인능정상표체. 결론 조직괴배양법괄합성기세포적원대배양;FIV재체능이고전염솔장AMF기인전지대서성기세포,병획득고효적표체.해방법 위일충교이상적AMF기인전염모식.
Objective To explore a safe and high efficiency way of gene transfection of autocrine motility factor(AMF) in order to provide experimental basis for transplantation of myoblasts carrying AMF gone. Methods Sprague Dawley rat myoblasts were cultured, purified, proliferated and immunohisto-chemically verified. Then, the myoblasts were transfected with AMF and eGFP (enhanced green fluores-cent protein) gene by FIV (feline immunodeficiency virus). Fluorescence microscope and laser scanning confocal microscope were employed to detect eGFP so as to verify positive transfection rate. Expression of AMF was detected by immunohistochemical method. Results Myoblasts with 98% purity could he ob-tained after two weeks of primary culture and purification. Positive transfection rate reached 90.4% when MOI (multiplicity of infection) was 100 (P <0.01). The transfected AMF gene could express normally. Conclusions Explant culture is a proper way in rat myoblast culture. Meanwhile, AMF gene can he effectively transfected into rat myoblast and well expressed via FIV.