液压转基因技术应用于大鼠再生肝转基因实验

Hydrodynamics-based transgene directively into rat regenerating liver in vivo

目的探讨液压转基因技术（HDT）应用于大鼠再生肝转基因的条件和方法。方法以2ml/s的速度将浓度为30mg/L的含目的基因的质粒注射入大鼠尾静脉,于注射前/后不同时间进行大鼠2/3肝切除（PH）,于PH后不同恢复时间称量大鼠体重（g）和再生肝重（g）,计算肝系数（Lc）,并从Lc±Lc＊0%、＊5%、＊10%、＊15%、＊20%、＊25%、＊30%、＊35%等15组中找出最佳组,作为计算不同恢复时间再生肝最适注射质粒溶液量的校正系数（Trc）;取大鼠肝右叶中部组织制备冷冻切片,在波长488nm的荧光显微镜下观察、计数1万个细胞中的绿色荧光蛋白阳性细胞百分率。结果PH后注射生理盐水和注射空质粒对肝再生的影响与对照（只进行PH）相比无显著差异。PH前液压转基因的合适时间是PH前≥12h;PH后所有时间均可进行液压转基因。PH后对肝再生大鼠进行液压转基因的转基因溶液体积为大鼠体重（g）×9%×1/3×相应的校正系数（Trc）。转入基因在体内的表达时间和丰度既受载体影响,又受插入的目的基因影响。结论液压转基因技术亦可有效地应用于大鼠再生肝转基因研究。

Objective To study the conditions and methods of hydrodynamics-based transgene into rat regenerating liver in vivo. Methods The solution with concentration 30mg/L gene-containing plasmid was injected into rat tail veins at a speed of 2ml/s, then partial hepateetomy （PH） was performed at different times before/after injection, finally the rat （g） and regenerating liver （g） were weighed, and the liver coefficient （Lc） was calculated. Out of 15 groups which are Lc Lc ＊ 0% , ＊ 5% , 10% , ＊ 15% , ＊ 20% , ＊ 25% , ＊ 30% , ＊ 35% , the most suitable group was chosen as correction coefficient to calculate the most appropriate volume of plasmid solution which was injected into the regenerating liver at different recovery times, and at the same time, right lobe of liver was gathered to make frozen section, then observe and quantify the positive green fluorescent protein （GFP） rate at 488 nm excitation wavelength. Results Injection of either physiological saline or empty plasmid has no significant difference compared with control （only PH performance）. The appropriate time of hydrodynamics-based transgene is more than 12 hours before PH or anytime after PH. The solution volume of hydrodynamics-based transgene into liver regenerating rat after PH is rat weight （g） × 9% × 1/3 × corresponding correction coefficient （Trc）. Both vector and target gene have effect on the time and abundance of gene expression. Conclusion Hydrodynamics-based transgene can effectively be applied to gene transfection in rat regenerating liver.