Machens H G, Morgan J R, Berthiaume F, Stefanovich P, Berger A C
Klinik für Plastische, Hand- und Wiederherstellungschirurgie, Medizinischen Hochschule Hannover.
Langenbecks Arch Chir Suppl Kongressbd. 1998;115(Suppl I):681-7.
Gene therapy was tested for inducing functional angiogenesis in the superficial rat epigastric island flap to allow earlier pedicle division. Autologous rat fibroblasts were grown, harvested, cultured and retrovirally transfected to produce PDGF-AA, an angiogenetically active protein. Stable gene expression was monitored by PDGF-AA ELISA. 180 animals were divided into 3 groups (I-III) and a bilateral flap created in each animal. In all experiments, the rightsided flap was subjected to experimental treatment and the left-sided flap served as control (1 ml saline 0.9%). During flap elevation, group I received 5 x 10(6) GMFB (genetically modified fibroblasts) plus 1 ml DMEM as medium. Group II was treated with 5 x 10(6) NMFB (non modified fibroblasts) plus 1 ml medium and group III received 1 ml medium alone. The flaps were sutured back and the vascular pedicle was bilaterally ligated and divided in each 10 animals during the following 6 days. 7 days later, the flaps were harvested, the amount of necrosis measured and histologically examined. The GMFB produced up the 560-times more PDGF-AA than the NMFB, measured by ELISA. The GMFB-treated flaps tolerated surgical division of the vascular pedicle significantly earlier than groups II and III. Histologically, fibroblasts persisted in all flaps of groups I and II without major inflammatory reaction. In all GMFB-treated flaps, massive angiogenesis could be demonstrated. By means of retroviral gene transfer autologous rat fibroblasts can be genetically modified for stable expression of the PDGF-A gene to produce high amounts of PDGF-AA, which is angiogenetically active. After injection into the panniculus carnosus, these cells induce functional angiogenesis to permit earlier division of the vascular pedicle in this flap model.
在大鼠腹壁浅岛状皮瓣中测试基因疗法以诱导功能性血管生成，从而允许更早地离断蒂部。培养、收获、培养自体大鼠成纤维细胞，并通过逆转录病毒转染以产生血管生成活性蛋白血小板衍生生长因子-AA（PDGF-AA）。通过PDGF-AA酶联免疫吸附测定法监测稳定的基因表达。将180只动物分为3组（I-III组），每组动物均制作双侧皮瓣。在所有实验中，右侧皮瓣接受实验性治疗，左侧皮瓣作为对照（1ml 0.9%生理盐水）。在皮瓣掀起过程中，I组接受5×10⁶个基因修饰的成纤维细胞（GMFB）加1ml杜氏改良 Eagle培养基（DMEM）作为培养基。II组用5×10⁶个未修饰的成纤维细胞（NMFB）加1ml培养基进行治疗，III组仅接受1ml培养基。将皮瓣缝合回原处，在接下来的6天里，每10只动物双侧结扎并离断血管蒂。7天后，取下皮瓣，测量坏死量并进行组织学检查。通过酶联免疫吸附测定法测得，GMFB产生的PDGF-AA比NMFB多560倍。GMFB处理的皮瓣比II组和III组能显著更早地耐受血管蒂的手术离断。组织学检查显示，I组和II组的所有皮瓣中均有成纤维细胞存留，且无明显炎症反应。在所有GMFB处理的皮瓣中，均可证实有大量血管生成。通过逆转录病毒基因转移，可对自体大鼠成纤维细胞进行基因修饰，使其稳定表达PDGF-A基因，从而产生大量具有血管生成活性的PDGF-AA。将这些细胞注射到肌膜后，可诱导功能性血管生成，从而允许在此皮瓣模型中更早地离断血管蒂。
