Nagy J A, Shockley T R, Masse E M, Harvey V S, Hoff C M, Jackman R W
Department of Pathology, Beth Israel Hospital, Boston, MA 02215, USA.
Gene Ther. 1995 Aug;2(6):402-10.
To evaluate the ability of genetically modified peritoneal mesothelial cells to deliver recombinant proteins to the systemic circulation, we used our previously described mesothelial cell-based ex vivo gene therapy strategy. Rat primary peritoneal mesothelial cells, isolated from parietal peritoneum by enzymatic digestion, were stably transfected (using strontium phosphate DNA co-precipitation) with the plasmid pSVTKgh to express a secreted reporter gene product, human growth hormone (hgh). Such hgh-secreting mesothelial cells were reseeded on the denuded peritoneal surface of syngeneic recipients and delivery of the reporter gene product to the systemic circulation was monitored by analysis of serum samples for the presence of hgh at various times after mesothelial cell implantation. Polymerase chain reaction (PCR) analysis demonstrated that the hgh-transfected mesothelial cells repopulated the denuded areas and remained attached there for at least 12 weeks. Moreover, these genetically modified mesothelial cells continued to express the reporter gene product in vivo and secreted hgh in sufficient quantity to be detected in the systemic circulation (ie statistically significant amounts of hgh could be measured in the serum of cyclosporine A-treated rats for at least 2 months; Mann-Whitney test, P < 0.05). Our results demonstrate the successful, sustained, systemic delivery of a recombinant protein by genetically modified peritoneal mesothelial cells following their reattachment to the peritoneal surface, and suggest the potential of ex vivo mesothelial cell-mediated gene therapy for the treatment of inherited or acquired disorders requiring delivery of therapeutic proteins to the circulation.
为了评估基因修饰的腹膜间皮细胞向体循环递送重组蛋白的能力，我们采用了我们先前描述的基于间皮细胞的离体基因治疗策略。通过酶消化从壁腹膜分离的大鼠原代腹膜间皮细胞，用质粒pSVTKgh进行稳定转染（使用磷酸锶DNA共沉淀法）以表达一种分泌型报告基因产物——人生长激素（hgh）。将这种分泌hgh的间皮细胞重新接种到同基因受体的剥脱腹膜表面，并通过分析间皮细胞植入后不同时间血清样本中hgh的存在情况来监测报告基因产物向体循环的递送。聚合酶链反应（PCR）分析表明，hgh转染的间皮细胞重新填充了剥脱区域，并在那里附着至少12周。此外，这些基因修饰的间皮细胞在体内继续表达报告基因产物，并分泌足够量的hgh以便在体循环中检测到（即在环孢素A处理的大鼠血清中至少2个月能检测到统计学上显著量的hgh；Mann-Whitney检验，P < 0.05）。我们的结果表明，基因修饰的腹膜间皮细胞重新附着到腹膜表面后能成功、持续地向体循环递送重组蛋白，并提示离体间皮细胞介导的基因治疗在治疗需要向循环系统递送治疗性蛋白的遗传性或获得性疾病方面具有潜力。
