虚血性心疾患に対するマクロファージを用いた低侵襲性遺伝子導入法の開発

Less Invasive Gene Therapy using Macrophages for Ischemic Heart Disease

要旨　虚血性心疾患に対する既存の遺伝子治療法に代わりうる方法として，ゼラチンにより遺伝子を導入したマクロファージを用いた新たな低侵襲性遺伝子導入法を試みた．ゼラチンに緑色蛍光蛋白遺伝子を結合させ，ゼラチン—遺伝子複合体を作製した．ラット腹腔マクロファージをこのゼラチン—遺伝子複合体とともに14日間培養したところ，細胞内に緑色蛍光蛋白の蛍光を認めた．次に12匹のラットを麻酔下に開胸し，左冠動脈前下行枝を180分間結紮後再灌流させた．再灌流直後にそのマクロファージを陰茎背静脈から投与し，60分後に心臓を摘出し免疫組織学的に検討した．緑色蛍光蛋白抗原陽性のマクロファージが心筋非虚血部よりも虚血部に多く認められた．このように，マクロファージへの遺伝子導入はゼラチンー遺伝子複合体により可能であり，マクロファージは末梢静脈より投与しても心筋虚血部へ移行したので，本方法の遺伝子導入法としての有用性が示唆された．

　We have developed a new gene-targeting method using macrophages. Positively charged lattice structures of gelatin combine with negatively charged DNA. We aimed at targeting genes towards jeopardized myocardial tissue using macrophages which had been transfected with gelatin-DNA complex and had been introduced into the peripheral vein. The gelatin was impregnated with DNA-encoding green fluorescent protein (GFP). The GFP-gene was transfected into rat peritoneal macrophages by means of co-culturing with gelatin-GFP gene complex over a period of 14 days. Anesthetized rats were subjected to 180 minutes of ligation of the left anterior descending coronary artery. and the transfected macrophages were injected into the superficial dorsal vein of the penis, followed by 60 minutes of reperfusion. The heart was removed, and extrinsic macrophages were analyzed with light microscopy. GFP expression was confirmed in 30.2±5.1% of macrophages as evidenced by FACS analysis. Immunohistochemical analyses revealed that GFP-antigen-positive cells existed in the jeopardized myocardium rather than in normal regions. Thus, macrophages transfected by gelatin-DNA complex administered through a peripheral vein were able to target jeopardized myocardial tissue in rats. The present system has advantages in that there is less invasiveness, a higher transfection rate, and more tissue targeting ability.