Development of Gene Delivery Systems to Intestinal Epithelial Cells as a Target

本研究采用双细胞培养系统，研究了转染小鼠或人IFN- β表达质粒后外源表达的干扰素- β在四种上皮细胞中的分泌极性：人结直肠癌Caco-2、小鼠鳞状细胞癌pamt、犬Mardin-Darby肾MDCK和猪小管上皮细胞LLC-PK1 i_1在前三种细胞中，瞬时表达的ifn - β主要从转染的细胞膜侧分泌；如果质粒应用于上或下隔室，ifn - β分别从顶膜或基底外膜分泌。有趣的是，从两侧同时转染，顶部是小鼠ifn - β基因，底部是人lfn - β基因，导致小鼠ifn - β分泌到上室，人ifn - β分泌到下室，反之亦然。lc - pk_1细胞呈非极化瞬时分泌。同时，从转染了ifn - β表达质粒的细胞系中建立的转化子的稳定分泌是非极化的。综上所述，这些结果表明上皮细胞对同一蛋白质有不同的蛋白质分选-分泌途径。分类方式似乎取决于蛋白质的表达方式，组成或融合。这些发现将有助于构建肠上皮细胞和其他极化细胞的基因转移策略。

In the present study, the secretion polarity of interferon (IFN)-beta exogenously expressed following transfection of mouse or human IFN-beta expression plasmid in four kinds of epithelial cells was studied using a bicameral culture system : human colorectal carcinoma Caco-2, mouse squamous cell carcinoma Pam-T, Mardin-Darby canine kidney MDCK, and porcine tubular epithelial cell LLC-PK1 I_n the former three kinds, transiently expressed IFN-beta was predominantly secreted from the cell membrane side to which the transfection was carried out ; if the plasmid was applied to the upper or lower compartment, IFN-beta was secreted from the apical or basolateral membrane, respectively. Interestingly, simultaneous transfection from both sides, apically with mouse IFN-beta gene and basally with human LFN-betag ene, resulted in mouse IFN-beta secretion into upper compartments and human IFN-beta secretion into lower compartments, and vice versa. LLC-PK_1 cells showed non-polarized transient secretion. Meanwhile, stable secretion from the transformants, which is established from either cell line transfected with IFN-beta expression plasmid was non-polarized. Taken together these results suggest that epithelial cells have various protein sorting-secretion pathways for the same protein. The sorting manner seems to be depended on how the protein is expressed, constitutive oremergent. These findings would be useful to construct the strategy for gene transfer into intestinal epithelial cells and other polarized cells.