液压转基因技术用于脂肪肝大鼠的肝脏转基因实验

Application of hydrodynamics-based transgene to gene transfection in rat fatty liver

目的探讨液压转基因技术(HDT)应用于大鼠脂肪肝转基因的条件、方法。方法以不同速度将不同体积、浓度的绿色荧光蛋白基因质粒pEGFP-C1一次性注射到脂肪肝大鼠尾静脉,于注射后不同时间取大鼠(各4只)各肝叶制备冷冻切片,在波长488nm的荧光显微镜下观察、计数各肝叶绿色荧光蛋白阳性细胞。结果在pEGFP-C1浓度为33mg/L、注射速度为2ml/s、注射液体积为大鼠体重的8.5%条件下,注射质粒后6h,各肝叶绿色荧光蛋白阳性细胞比例最高,其中,蒂状叶的绿色荧光蛋白阳性细胞约占18%,左叶约占14%、中叶约占12.5%、右叶约占10%,尾状叶约占8%。转基因后24h绿色荧光蛋白的表达量逐渐减少,至72h时各肝叶均难以检出绿色荧光蛋白阳性细胞。结论大鼠尾静脉液压转基因技术可用于脂肪肝大鼠的肝脏转基因研究,转基因的适宜条件为:质粒溶液浓度33mg/L,注射量占大鼠体重的8.5%,注射速度为2ml/s,观察转基因效果的适宜时间是转基因后6～24h。

Objective To study the condition and method of hydrodynamics-based transgene（HDT） in rat fatty liver. Methods Inject different dosages and concentrations of green fluorescent protein plasmid pEGFP-C1 at different speeds, then collect 4 rats＇ liver leaves at different time points after injection and prepare their frozen section, finally observe and quantify the GFP expression with fluorescence microscope at 488 nm excitation wavelength. Results Plasmid pEGFP-C1 concentration 33mg/L, injection speed 2ml/s, injection volume 8.5% of rat body weight, injected plasmid. After 6 hours of injection, GFP-positive cells rate of pedicel leaf is about 18% , left leaf about 14% , middle leaf about 12.5% , right leaf about 10% and tail leaf about 8%. GFP begin to gradually reduce since 24 hours, until 72 hours almost no GFP-positive ceils were checked in all liver leaves. Conclusion Hydrodynamics-based transgene can be applied to rat fatty liver, the appropriate conditions of this method are 33mg/L plasmid concentration, 8.5% rat avoirdupois, 2ml/s injection speed, and the suitable time to observe the proportion of GFP-positive cells is 6-24 hours after gene injection.