不同转染途径对慢病毒载体基因在大鼠肝脏中表达的影响

Effect of different approaches of lentiviral vector transfection on target gene expression in rat liver

目的研究慢病毒载体基因在肝脏内转染的有效途径。方法本实验根据大鼠消化系统解剖,设计利用经回盲部回结肠静脉穿刺置管至门静脉作为转染途径。通过观察对比尾静脉转染途径大鼠肝脏荧光蛋白表达、基因表达及转氨酶变化情况,探讨不同转染方法的有效性和安全性。结果门静脉转染组和尾静脉转染组大鼠均存活。转染后96 h慢病毒载体携带绿色荧光蛋白在门静脉转染组肝脏呈高表达,14 d后仍有持续表达;尾静脉转染组术后96 h荧光蛋白表达较门静脉组弱,14 d后几无持续表达。RT-PCR检测转染基因LXRα-RNAi效率:转染组LXRαmRNA表达均有下调,门静脉转染组LXRα基因敲减率明显高于尾静脉转染组(0.135±0.002 vs 0.713±0.036,P<0.05),两组血清ALT比较差别无统计学意义。结论经门静脉灌注是基因转染的有效途径,经回盲部回结肠静脉属支穿刺置管可在保证大鼠存活的情况下提高基因转染率,且对肝功能无明显损害,是值得推广的方法。

Objective To investigate the optimal approach of lentiviral vector transfection for effective delivery of exogenous gene into the liver. Methods The lentiviral vector was delivered via the ileocolic vein of the ileocecus （portal vein group） or via the caudal vein of SD rats. The effect gene transfection into the liver was assessed by observing the expression of green fluorescence protein expression carried by the lentiviral vector, silencing of LXRa mRNA expression mediated by RNA interference, and liver transaminase changes. The efficiency and safety of the two approaches of transfection were evaluated. Results All the rats receiving lentiviral transfection survived. In the portal vein group, abundant green fluorescence was detected in the liver at 96 h following the trasnfection and lasted till 14 days, whereas only weak fluorescence was observed in the caudal vein group. The results of RT-PCR demonstrated a significant higher rate of LXRa knock-down in portal vein group than in caudal vein group （0.135±0.002 vs 0.713±0.036, P〈0.05）. No significant difference in ALT levels found between the two groups. Conclusion Infusion via the potal vein is effective for gene transfection into the liver, and puncture from the ileocolic vein of ileocecus can guarantee the survival of rats and improve the transfection efficiency without causing liver injury.