Powell J T, Klaasse Bos J M, van Mourik J A
CLB, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
FEBS Lett. 1992 Jun 1;303(2-3):173-7. doi: 10.1016/0014-5793(92)80512-f.
The conditions and efficacy of transfection of vascular cells in primary culture using DEAE-dextran, calcium phosphate and lipofectin have been investigated using chloramphenicol acetyltransferase and luciferase as reporter genes. Subsequently factor VIII was expressed in endothelial and smooth muscle cells. Both reporter genes could be expressed after transfection of umbilical vein endothelial cells, umbilical artery smooth muscle cells and fibroblasts. The expression of both reporter genes in endothelial and smooth muscle cells was highest using lipofectin. After transfection of smooth muscle cells with both full-length and mutant factor VIII genes, factor VIII activity and antigen were secreted into the culture medium, the secretion remaining stable to serial cell passage. The secretion of factor VIII from transfected smooth muscle cells was confirmed by the immunoprecipitation of [35S]methionine labelled protein. Endothelial cells also were successfully transfected with the mutant factor VIII gene.
以氯霉素乙酰转移酶和荧光素酶作为报告基因，研究了用二乙氨基乙基葡聚糖、磷酸钙和脂质体对原代培养的血管细胞进行转染的条件和效果。随后，因子Ⅷ在内皮细胞和平滑肌细胞中得以表达。在转染脐静脉内皮细胞、脐动脉平滑肌细胞和成纤维细胞后，两种报告基因均可表达。在内皮细胞和平滑肌细胞中，使用脂质体时两种报告基因的表达最高。在用全长和突变型因子Ⅷ基因转染平滑肌细胞后，因子Ⅷ活性和抗原分泌到培养基中，并且在连续传代细胞中该分泌保持稳定。通过对[35S]甲硫氨酸标记的蛋白质进行免疫沉淀，证实了转染的平滑肌细胞分泌因子Ⅷ。突变型因子Ⅷ基因也成功转染了内皮细胞。
