Pickering J G, Jekanowski J, Weir L, Takeshita S, Losordo D W, Isner J M
Department of Medicine (Cardiology), St Elizabeth's Hospital, Tufts University School of Medicine, Boston, Mass. 02135.
Circulation. 1994 Jan;89(1):13-21. doi: 10.1161/01.cir.89.1.13.
Complexing recombinant DNA with cationic liposomes is a convenient means of introducing foreign genes into cells (lipofection) and could potentially form the basis for genetically modifying diseased blood vessels in patients. The mechanism of lipofection is incompletely understood, but it is recognized that the degree of successful gene transfer is highly dependent on cell type. To date, there has been no reported experience with lipofection of human vascular smooth muscle cells.
Primary cultures of human vascular smooth muscle cells were transfected under optimized conditions with a plasmid expressing either firefly luciferase (Luc) or nuclear-localized beta-galactosidase (NL-beta-gal). Cells were derived from either normal human internal mammary arteries (n = 6), fragments of primary atherosclerotic plaque (n = 4), or fragments of restenotic lesions (n = 5). Concurrent lipofection of rabbit vascular smooth muscle cells and NIH 3T3 cells was performed as well. Cultures derived from 15 patients all demonstrated positive expression of the reporter gene. Compared with NIH 3T3 cells, however, expression in human vascular smooth muscle cells was markedly reduced: in cells derived from internal mammary artery, Luc expression, normalized for protein content, was 123-fold lower than in NIH 3T3 cells, whereas the proportion of cells expressing NL-beta-gal was 30-fold lower. Luc expression in cells derived from restenotic tissue was significantly greater than from cells derived from primary plaque (P < .03). Within a given population of cells, the mitotic index of cells expressing the recombinant gene was significantly higher than the mitotic index for the total population of cells (P < .05). Finally, cotransfection experiments, in which lipofection of smooth muscle cells was performed using genes for NL-beta-gal and for human growth hormone, showed that among positive transfects, a high proportion of cells (23% to 36%) coexpressed both genes.
The efficiency of successful lipofection in human vascular smooth muscle cells in vitro is low. Transfection appears to be preferentially facilitated in cells derived from restenotic tissue, and specific properties of smooth muscle cells, including growth rates, appear to be critical for successful transfection. Further elucidation of cell properties that promote transfection is required to augment the efficiency of liposome-mediated gene transfer in human vascular cells.
将重组DNA与阳离子脂质体复合是将外源基因导入细胞的一种便捷方法（脂质体转染），并且有可能成为对患者病变血管进行基因改造的基础。脂质体转染的机制尚未完全明了，但人们认识到基因转移成功的程度高度依赖于细胞类型。迄今为止，尚无关于人血管平滑肌细胞脂质体转染的报道。
在优化条件下，用表达萤火虫荧光素酶（Luc）或核定位β-半乳糖苷酶（NL-β-gal）的质粒转染人血管平滑肌细胞原代培养物。细胞来源于正常人乳内动脉（n = 6）、原发性动脉粥样硬化斑块碎片（n = 4）或再狭窄病变碎片（n = 5）。同时也对兔血管平滑肌细胞和NIH 3T3细胞进行了脂质体转染。来自15名患者的培养物均显示报告基因的阳性表达。然而，与NIH 3T3细胞相比，人血管平滑肌细胞中的表达明显降低：在来源于乳内动脉的细胞中，以蛋白质含量标准化后的Luc表达比NIH 3T3细胞低123倍，而表达NL-β-gal的细胞比例低30倍。再狭窄组织来源的细胞中的Luc表达明显高于原发性斑块来源的细胞（P <.03）。在给定的细胞群体中，表达重组基因的细胞的有丝分裂指数明显高于细胞总群体的有丝分裂指数（P <.05）。最后，共转染实验中，使用NL-β-gal和人生长激素基因对平滑肌细胞进行脂质体转染，结果显示在阳性转染细胞中，高比例的细胞（23%至36%）同时表达这两种基因。
体外人血管平滑肌细胞中成功脂质体转染的效率较低。转染似乎在再狭窄组织来源的细胞中更容易实现，并且平滑肌细胞的特定特性，包括生长速率，似乎对成功转染至关重要。需要进一步阐明促进转染的细胞特性，以提高脂质体介导的人血管细胞基因转移效率。
