Lecocq Michèle, Andrianaivo Fanjam, Warnier Marie-Thérèse, Wattiaux-De Coninck Simone, Wattiaux Robert, Jadot Michel
Laboratoire de Chimie Physiologique, Facultés Universitaires Notre-Dame de la Paix, 61 rue de Bruxelles, 5000 Namur, Belgium.
J Gene Med. 2003 Feb;5(2):142-56. doi: 10.1002/jgm.328.
An efficient gene transfer can be achieved in mouse liver by a rapid tail vein injection of a large volume of plasmid DNA solution (hydrodynamics-based transfection). The mechanism of gene transfer by this procedure is not known. It must be related to the uptake and intracellular fate of DNA.
We have investigated the problem by following the uptake by mouse liver and the intracellular distribution of DNA after a rapid tail vein injection of a large (2.0 ml) or a small (0.2 ml) volume of (35)S-DNA solution. Total and acid-soluble radioactivity were measured in liver homogenates at increasing times after injection, and their subcellular distributions were established by centrifugation methods and compared with the distributions of marker enzymes of the membrane compartments involved in endocytosis: alkaline phosphodiesterase (plasma membrane) and cathepsin C (lysosomes).
(35)S-DNA uptake by the liver is similar when a small or a large volume of injection is used but its degradation is markedly slower after a 2.0 ml injection. When a small volume of injection is given, distribution of radioactivity after differential centrifugation indicates that the plasmid DNA is endocytosed and reaches lysosomes where it is hydrolysed. After a large volume injection, part of (35)S-DNA has the same fate, another part remains acid-precipitable for at least 1 h and is associated with structures sedimenting at low centrifugation speed in the nuclear fraction N. Analysis of that fraction by gradient centrifugation suggests that these structures are plasma membrane fragments that could originate from the apical domain of hepatocytes. The proportion of (35)S-DNA associated with hepatocytes is about doubled after a large volume injection. Fractionation of isolated hepatocytes by centrifugation confirms results obtained on the whole liver. Treatment of the N fraction or isolated hepatocytes with pancreatic DNAse illustrates that (35)S-DNA that remains bound to plasma membrane after a large volume injection is located on the outer face.
The fact that after an hydrodynamic injection (35)S-DNA remains bound to the outside face of the plasma membrane for at least 1 h indicates that it is not, or very slowly, internalised during that period. The relatively small difference in the amount of DNA picked up by hepatocytes depending on the type of injection could not explain the absence of expression after a conventional injection and the strong expression after a hydrodynamic injection. If DNA enters the cells by endocytosis, even after an hydrodynamic injection, its persistence at the outside face of the plasma membrane could favour transfection by allowing hepatocytes to dispose for a relatively long time of a reservoir of intact DNA.
通过快速尾静脉注射大量质粒DNA溶液（基于流体动力学的转染）可在小鼠肝脏中实现高效基因转移。该方法的基因转移机制尚不清楚。它必定与DNA的摄取及细胞内命运相关。
我们通过在快速尾静脉注射大体积（2.0 ml）或小体积（0.2 ml）的（35）S-DNA溶液后追踪小鼠肝脏对其的摄取及DNA的细胞内分布来研究该问题。在注射后不同时间测量肝脏匀浆中的总放射性和酸溶性放射性，并通过离心方法确定其亚细胞分布，同时与参与内吞作用的膜区室的标记酶——碱性磷酸二酯酶（质膜）和组织蛋白酶C（溶酶体）的分布进行比较。
无论注射体积大小，肝脏对（35）S-DNA的摄取相似，但在注射2.0 ml后其降解明显更慢。当注射小体积溶液时，差速离心后的放射性分布表明质粒DNA被内吞并到达溶酶体，在那里被水解。大体积注射后，部分（35）S-DNA有相同命运，另一部分至少1小时内仍可被酸沉淀，并与核组分N中低速离心时沉淀的结构相关。通过梯度离心对该组分进行分析表明，这些结构是可能源自肝细胞顶端区域的质膜片段。大体积注射后与肝细胞相关的（35）S-DNA比例约增加一倍。通过离心对分离的肝细胞进行分级分离证实了在整个肝脏上获得的结果。用胰DNA酶处理N组分或分离的肝细胞表明，大体积注射后仍与质膜结合的（35）S-DNA位于外表面。
流体动力学注射后（35）S-DNA至少1小时内仍与质膜外表面结合这一事实表明在此期间它未被内化或内化非常缓慢。根据注射类型，肝细胞摄取的DNA量相对较小的差异无法解释常规注射后无表达而流体动力学注射后有强表达的现象。如果DNA通过内吞作用进入细胞，即使在流体动力学注射后，其在质膜外表面的持续存在可能通过使肝细胞在相对较长时间内有完整DNA储备而有利于转染。
