Knoll Thomas, Sagi Sreedhar, Trojan Lutz, Schaaf Axel, Alken Peter, Michel Maurice Stephan
Department of Urology, University Hospital Mannheim, 68135 Mannheim, Germany.
Urol Res. 2004 May;32(2):129-32. doi: 10.1007/s00240-003-0395-1. Epub 2004 Jan 13.
Cystinuria is the cause of 1-2% of stones observed in adults and about 10% of those occurring in children. Recurrent stone formation and multiple operations cause considerable morbidity. We investigated the transfection efficiency of naked plasmid DNA in porcine kidney cells by applying holmium laser (Ho:YAG) energy in vitro as well as ex vivo in a porcine kidney papilla model. In the in vitro experiments, naked plasmid DNA was added to LLC-PK1 cells suspended in a medium and Ho:YAG laser applied with varying pulses. The transfection efficiency was measured by the expression of EGFP reporter gene in the cells by FACScan analysis and fluorescence microscopy. Ex vivo, papilla from porcine kidney was excised and naked plasmid DNA was added to the tissue in the medium. The laser was then applied and the cryosectioned tissue observed under fluorescence microscope. The efficiency of transfection in vitro significantly improved with the increase in impulses (P<0.01). Transfection at 50 impulses averaged 0.7+/-0.3%, at 200 impulses 28.3+/-7.7%, and at 500 impulses 36.1+/-3.1%. The cell mortality rate increased with higher pulse rate up to 70%. Ex vivo trials showed transfection in extended regions of the tissue and also in the peripheral layers of the papilla. Our study indicates that the transfection of benign kidney cells by Ho:YAG is a promising new gene transfer strategy. The ex vivo trials showed that peripheral renal tissue layers are susceptible to transfection by Ho:YAG applied from the papillary surface.
胱氨酸尿症是成人中1%-2%的结石以及儿童中约10%的结石的病因。复发性结石形成和多次手术会导致相当高的发病率。我们通过在体外以及在猪肾乳头模型中进行离体实验，应用钬激光（Ho:YAG）能量来研究裸质粒DNA在猪肾细胞中的转染效率。在体外实验中，将裸质粒DNA添加到悬浮在培养基中的LLC-PK1细胞中，并以不同脉冲施加Ho:YAG激光。通过FACScan分析和荧光显微镜观察细胞中EGFP报告基因的表达来测量转染效率。在离体实验中，切除猪肾乳头，将裸质粒DNA添加到培养基中的组织中。然后施加激光，并在荧光显微镜下观察冷冻切片组织。体外转染效率随着脉冲数的增加而显著提高（P<0.01）。50次脉冲时的转染率平均为0.7±0.3%，200次脉冲时为28.3±7.7%，500次脉冲时为36.1±3.1%。细胞死亡率随着脉冲率的升高而增加，最高可达70%。离体实验表明，在组织的扩展区域以及乳头的外周层均有转染。我们的研究表明，Ho:YAG对良性肾细胞的转染是一种有前景的新基因转移策略。离体实验表明，从乳头表面施加Ho:YAG时，肾外周组织层易于被转染。
