Aleksic I, Ren M, Czer L S, Freimark D, Dalichau H, Blanche C, Trento A, Barath P
Division of Cardiothoracic Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
Thorac Cardiovasc Surg. 1996 Apr;44(2):81-5. doi: 10.1055/s-2007-1011991.
Rejection remains a major problem after cardiac transplantation. One hypothesis is that transfer of recipient HLA genes could lead to expression of the antigens on the surface of donor cells and so facilitate graft acceptance. This paper describes a pilot study for relevant gene-transfer (transfection) experiments on adult cardiac myocytes, investigating the feasibility of transfection using cationic liposomes. The plasmid pcDV1-pL2 vector containing HLA-DR-alpha chain gene was incubated with Lipofectin, a DOTMA and DOPE lipid mixture, for 10 minutes. Embryonic chick cardiac myocytes (ECCM) and COS7 monkey cells were incubated with DNA: Lipofectin ratios of 1:1, 1:2, 1:4, and 1:10 for 16 hours (hrs). Using a fixed ratio of 1:4, incubation periods of 4, 8, and 16 hrs were compared. Finally, cells were incubated for 16 hrs and consecutively cultured for 6 days. Expression of the HLA-DR-alpha chain antigen was detected by indirect immunohistochemical staining. Highest transfection rates were achieved with a DNA: Lipofectin ratio of 1:4 for ECCM and COS7 cells (2.7% +/- 0.6% and 2.4% +/- 0.3% after 16 hrs incubation) and a transfection time of 4 hrs (5.8% +/- 0.6% and 5.3% +/- 1.7%). Immunohistochemically positive cells were present after 6 days (2.0% +/- 1.2% and 2.1% +/- 0.3%). We found a low level of expression of HLA-DR-alpha chain gene, influenced by transfection time and DNA: Lipofectin ratio. To increase the efficiency of liposome-mediated gene transfer and assess potential applications in cardiac transplantation, further investigation of cell properties promoting transfection is necessary.
心脏移植后排斥反应仍是一个主要问题。一种假说认为，受者HLA基因的转移可导致供体细胞表面抗原的表达，从而促进移植物的接受。本文描述了一项针对成年心肌细胞进行相关基因转移（转染）实验的初步研究，探讨使用阳离子脂质体进行转染的可行性。将含有HLA-DR-α链基因的质粒pcDV1-pL2载体与Lipofectin（一种DOTMA和DOPE脂质混合物）孵育10分钟。将胚胎鸡心肌细胞（ECCM）和COS7猴细胞分别以DNA与Lipofectin 1:1、1:2、1:4和1:10的比例孵育16小时。使用1:4的固定比例，比较4小时、8小时和16小时的孵育时间。最后，将细胞孵育16小时并连续培养6天。通过间接免疫组织化学染色检测HLA-DR-α链抗原的表达。对于ECCM和COS7细胞，DNA与Lipofectin比例为1:4时转染率最高（孵育16小时后分别为2.7%±0.6%和2.4%±0.3%），转染时间为4小时时转染率最高（分别为5.8%±0.6%和5.3%±1.7%）。6天后存在免疫组织化学阳性细胞（分别为2.0%±1.2%和2.1%±0.3%）。我们发现HLA-DR-α链基因的表达水平较低，受转染时间和DNA与Lipofectin比例的影响。为了提高脂质体介导的基因转移效率并评估其在心脏移植中的潜在应用，有必要进一步研究促进转染的细胞特性。
