携带自分泌运动因子基因的慢病毒载体转染大鼠成肌细胞

Transfection of rat myoblasts with ieutivirus carrying autocrine motility factor gene

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（enhanced green fluorescent protein，EGFP）基因的猫免疫缺陷病毒（feline immunodeficiency virus，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（multiplicity of infection，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。

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 gene. Methods Sprague Dawley rat myoblasts were cultured, purified, proliferated and immunohistochemically verified. Then, the myoblasts were transfected with AMF and eGFP （enhanced green fluorescent protein） gene by FIV （fehne 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 be obtained after two weeks of primary culture and purification. Positive transfectian rate reached 90. 4% when MOI （multiplicity of infection） was 100 （P 〈0.01 ）. The transfected AMF gene could express normally. Condusions Explant culture is a proper way in rat myoblast culture. Meanwhile, AMF gene can be effectively transfected into rat myoblast and well expressed via FIV.