Jackman R W, Stapleton T D, Masse E M, Harvey V S, Meyers M S, Shockley T R, Nagy J A
Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.
Hum Gene Ther. 1998 May 1;9(7):1069-81. doi: 10.1089/hum.1998.9.7-1069.
We have used our previously described ex vivo mesothelial cell (MC)-mediated gene therapy strategy (Gene Ther. 2:393-401, 1995) to modify the functional properties of the rat parietal peritoneal mesothelium in vivo by expression of a membrane-bound recombinant protein on the MC surface. Rat primary MCs were stably transfected (using strontium phosphate DNA coprecipitation) with a plasmid containing the gene for rat thrombomodulin (TM), a transmembrane glycoprotein that functions as an essential cofactor for the physiological activation of the anticoagulant protein C by the enzyme thrombin. As demonstrated by immunohistochemistry and by direct equilibrium binding with radiolabeled thrombin, genetically modified MCs expressed high levels of TM antigen on their surface in vitro. As judged by a thrombin-dependent protein C activation assay, such MC membrane-bound TM was biologically active. Once reseeded on the denuded parietal peritoneal surface of syngeneic recipients, these TM-transfected MCs continued to express TM antigen in vivo for at least 90 days. Moreover, the recombinant TM expressed on the reconstituted parietal mesothelium retained its ability to activate protein C in a thrombin-dependent manner. Our data indicate that MC-mediated expression of TM can be used to augment the anticoagulant properties of the parietal peritoneal surface. In general, our results suggest that ex vivo MC-mediated gene therapy can be used to deliver other therapeutic transmembrane proteins to the MC surface to enhance the functional repertoire of the parietal mesothelium in vivo.
我们采用了我们之前描述的离体间皮细胞（MC）介导的基因治疗策略（《基因治疗》2:393 - 401，1995年），通过在MC表面表达一种膜结合重组蛋白，在体内改变大鼠壁层腹膜间皮的功能特性。大鼠原代MC用含有大鼠血栓调节蛋白（TM）基因的质粒（使用磷酸锶DNA共沉淀法）进行稳定转染，TM是一种跨膜糖蛋白，作为凝血酶将抗凝蛋白C生理激活的必需辅因子发挥作用。免疫组织化学以及与放射性标记凝血酶的直接平衡结合实验表明，基因修饰的MC在体外其表面表达高水平的TM抗原。通过凝血酶依赖性蛋白C激活试验判断，这种MC膜结合的TM具有生物活性。一旦重新接种到同基因受体剥脱的壁层腹膜表面，这些转染TM的MC在体内至少持续表达TM抗原90天。此外，在重建的壁层间皮上表达的重组TM保留了以凝血酶依赖性方式激活蛋白C的能力。我们的数据表明，MC介导的TM表达可用于增强壁层腹膜表面的抗凝特性。总体而言，我们的结果表明，离体MC介导的基因治疗可用于将其他治疗性跨膜蛋白递送至MC表面，以增强体内壁层间皮的功能储备。
