Saldeen J, Curiel D T, Eizirik D L, Andersson A, Strandell E, Buschard K, Welsh N
Department of Medical Cell Biology, Uppsala University, Sweden.
Diabetes. 1996 Sep;45(9):1197-203. doi: 10.2337/diab.45.9.1197.
The establishment of gene delivery systems that result in efficient transfection of the pancreatic beta-cells may generate an important tool for the study of IDDM and may also represent one critical step toward a clinical application of gene transfer for the prevention or early treatment of the disease. Using the reporter gene vectors pCAT and pCMV beta-gal, we have investigated the efficiency of transfection mediated by calcium phosphate precipitation, the monocationic liposome Lipofectin, the polycationic liposome Lipofectamine, and adenovirus-polylysine (AdpL) DNA complexes in human, mouse, rat, and fetal porcine islet cells. In all species studied, calcium phosphate-mediated transfection resulted in lower chloramphenicol acetyl transferase (CAT) activities than the other methods. Intact human, mouse, and rat islets were poorly transfected by Lipofectin, Lipofectamine, and AdpL. When dispersed by trypsin treatment, however, human, mouse, rat, and fetal pig islect cells were efficiently transfected by Lipofectamine. Moreover, transfection of dispersed human and mouse islet cells using AdpL, also resulted in high CAT activities. The percentage of cells staining positively for beta-galactosidase after transfection with Lipofectamine was 49% for mouse, 56% for rat, and 57% for dispersed human islet cells. Transfection of human islet cells using AdpL, however, yielded 70% beta-gal-positive cells. Fluorescence-activated cell sorting-purified rat islet alpha- and beta-cells were transfected with similar efficiency using Lipofectamine. CAT expression in human islet cells transfected with either Lipofectamine or AdpL reached a peak value after 5-7 days, followed by a gradual decline. It is concluded that transfection with AdpL or Lipofectamine are both efficient means to achieve transient expression of gene constructs in human and mouse islet cells, while for rat and fetal porcine islet cells, Lipofectamine is the most efficient of the agents investigated in this study.
建立能够有效转染胰腺β细胞的基因传递系统，可能会为胰岛素依赖型糖尿病（IDDM）的研究提供一个重要工具，也可能是朝着基因转移临床应用以预防或早期治疗该疾病迈出的关键一步。我们使用报告基因载体pCAT和pCMV β-半乳糖苷酶，研究了磷酸钙沉淀法、单阳离子脂质体Lipofectin、聚阳离子脂质体Lipofectamine以及腺病毒-聚赖氨酸（AdpL）DNA复合物在人、小鼠、大鼠和胎猪胰岛细胞中介导的转染效率。在所有研究的物种中，磷酸钙介导的转染产生的氯霉素乙酰转移酶（CAT）活性低于其他方法。完整的人、小鼠和大鼠胰岛被Lipofectin、Lipofectamine和AdpL转染的效率很低。然而，经胰蛋白酶处理分散后，人、小鼠、大鼠和胎猪胰岛细胞能被Lipofectamine高效转染。此外，使用AdpL转染分散的人及小鼠胰岛细胞，也能产生较高的CAT活性。用Lipofectamine转染后，β-半乳糖苷酶染色呈阳性的细胞百分比，小鼠为49%，大鼠为56%，分散的人胰岛细胞为57%。然而，用AdpL转染人胰岛细胞，β-半乳糖苷酶阳性细胞的比例为70%。用Lipofectamine转染经荧光激活细胞分选纯化的大鼠胰岛α细胞和β细胞，效率相似。用Lipofectamine或AdpL转染的人胰岛细胞中，CAT表达在5 - 7天后达到峰值，随后逐渐下降。结论是，AdpL或Lipofectamine转染都是在人及小鼠胰岛细胞中实现基因构建体瞬时表达的有效方法，而对于大鼠和胎猪胰岛细胞，Lipofectamine是本研究中所研究的试剂中最有效的。
