Sawa Y, Kadoba K, Suzuki K, Bai H Z, Kaneda Y, Shirakura R, Matsuda H
First Department of Surgery, Osaka University Medical School, Japan.
J Thorac Cardiovasc Surg. 1997 Mar;113(3):512-8; discussion 518-9. doi: 10.1016/S0022-5223(97)70364-7.
To confirm gene transfer techniques especially into the whole heart, we tried out a gene transfer method involving liposome with the viral envelope hemagglutinating virus of Japan liposome as an alternative to existing techniques such as cationic lipofection or other viral vectors.
For this study, hemagglutinating virus of Japan liposome (H group) or cationic liposome (L group) was used to compare the efficacy of gene transfection of oligonucleotide labeled with fluorescein isothiocyanate and cDNA of beta-galactosidase and human manganese-superoxide dismutase. Fluorescein-labeled oligonucleotide, cDNA of beta-galactosidase, or manganese-superoxide dismutase was complexed with liposomes, DNA-binding nuclear protein, and the viral protein coat of hemagglutinating virus of Japan. After donor rat hearts arrested by cardioplegia had been harvested, the coronary artery during cardioplegic arrest was infused via an aortic cannula with the liposome-gene complex. Next, the hearts were transplanted into the abdomen of recipient rats of the same strain, and all recipients were put to death after 3 days of transfection.
Fluorescein isothiocyanate was detected in the nuclei of more than 70% of the myocytes (75% +/- 14%, n = 5) in the H group compared with fewer than 10% in the L group (7% +/- 5%, n = 5). The intensity of fluorescein isothiocyanate was significantly higher in the H group (979 +/- 112 FI) than in the L group (116 +/- 68 FI). beta-Galactosidase was expressed in the cytosol of more than 50% of the myocytes in the H group (61% +/- 7%, n = 5) compared with none in the L group (0%, n = 5). After 3 days of gene transfection, and when exposed to ischemia (30 minutes, 37 degrees C) and reperfusion (30 minutes, 37 degrees C) with Langendorff apparatus, the hearts transfected with manganese-superoxide dismutase (S group, n = 5) showed a significantly higher percentage of recovery of left ventricular end-diastolic pressure (S vs C, 86% +/- 3% vs 54% +/- 12%) and coronary flow (98% +/- 2% vs 66% +/- 12%) than did the control hearts (C group, n = 5). Western blotting analysis showed an apparent increased expression of manganese-superoxide dismutase in the hearts transfected with manganese-superoxide dismutase compared with the control hearts. These results clearly demonstrated that the donor hearts were transfected with fluorescein-labeled oligonucleotide and the beta-galactosidase gene as a result of coronary infusion of the hemagglutinating virus of Japan liposome during cardioplegic arrest at the time of harvest. Furthermore, the hearts transfected with manganese-superoxide dismutase showed significant improvement in tolerance against ischemia reperfusion injury.
We believe that this method represents a novel in vivo gene transfer technique for the heart and thus may provide a new tool for research and therapy of heart transplantation.
为了确定基因转移技术，尤其是针对整个心脏的基因转移技术，我们尝试了一种基因转移方法，该方法涉及使用带有日本血凝病毒包膜的脂质体，以此替代诸如阳离子脂质转染或其他病毒载体等现有技术。
在本研究中，使用日本血凝病毒脂质体（H组）或阳离子脂质体（L组）来比较用异硫氰酸荧光素标记的寡核苷酸以及β-半乳糖苷酶和人锰超氧化物歧化酶的cDNA的基因转染效率。将异硫氰酸荧光素标记的寡核苷酸、β-半乳糖苷酶的cDNA或锰超氧化物歧化酶与脂质体、DNA结合核蛋白以及日本血凝病毒的病毒蛋白衣壳复合。在通过心脏停搏法使供体大鼠心脏停跳后取出心脏，在心脏停搏期间经主动脉插管向冠状动脉注入脂质体-基因复合物。接下来，将心脏移植到同品系受体大鼠的腹部，所有受体在转染3天后处死。
H组中超过70%的心肌细胞（75%±14%，n = 5）的细胞核中检测到异硫氰酸荧光素，而L组中少于10%（7%±5%，n = 5）。H组中异硫氰酸荧光素的强度（979±112 FI）明显高于L组（116±68 FI）。H组中超过50%的心肌细胞（61%±7%，n = 5）的细胞质中表达了β-半乳糖苷酶，而L组中无表达（0%，n = 5）。基因转染3天后，在用Langendorff装置进行缺血（30分钟，37℃）和再灌注（30分钟，37℃）处理时，转染了锰超氧化物歧化酶的心脏（S组，n = 5）的左心室舒张末期压力恢复百分比（S组与C组相比，86%±3%对54%±12%）和冠状动脉血流量（98%±2%对66%±12%）明显高于对照心脏（C组，n = 5）。蛋白质印迹分析显示，与对照心脏相比，转染了锰超氧化物歧化酶的心脏中锰超氧化物歧化酶的表达明显增加。这些结果清楚地表明，在收获时心脏停搏期间经冠状动脉注入日本血凝病毒脂质体后，供体心脏被异硫氰酸荧光素标记的寡核苷酸和β-半乳糖苷酶基因转染。此外，转染了锰超氧化物歧化酶的心脏对缺血再灌注损伤的耐受性有显著改善。
我们认为这种方法代表了一种用于心脏的新型体内基因转移技术，因此可能为心脏移植的研究和治疗提供一种新工具。
Zotero 插件