Machens H G, Morgan J R, Berthiaume F, Stefanovich P, Reimer R, Berger A C
Clinic for Plastic Hand and Reconstructive Surgery, Hannover Medical School, Germany.
Langenbecks Arch Surg. 1998 Oct;383(5):345-50. doi: 10.1007/s004230050146.
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 platelet-derived growth factor AA (PDGF-AA), an angiogenetically active protein. Stable gene expression was monitored by PDGF-AA enzyme-linked immunosorbent assay (ELISA). One hundred and eighty animals were divided into three groups (I-III) and a bilateral flap created in each animal. In all experiments, the right-sided flap was subjected to experimental treatment and the left-sided flap served as control (1ml saline 0.9%). During flap elevation, group I received 5X10(6) GMFB (genetically modified fibroblasts) plus 1 ml Dulbecco's modified Eagle's medium. Group II was treated with 5x10(6) NMFB (non-modified fibroblasts) plus 1 ml medium and group III received 1 ml medium only. The flaps were sutured back and the vascular pedicle was bilaterally ligated and divided in each of ten animals during the following 6 days. After 7 days, the flaps were harvested, the amount of necrosis measured and histologically examined.
The GMFB produced up to 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.
对基因疗法进行测试，以诱导大鼠腹壁浅岛状皮瓣实现功能性血管生成，从而能更早地离断蒂部。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使其产生血小板衍生生长因子AA（PDGF - AA），这是一种具有血管生成活性的蛋白质。通过PDGF - AA酶联免疫吸附测定（ELISA）监测稳定的基因表达。180只动物分为三组（I - III组），每只动物制作双侧皮瓣。在所有实验中，右侧皮瓣接受实验性处理，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GMFB（基因修饰的成纤维细胞）加1ml杜氏改良 Eagle培养基。II组用5×10⁶个NMFB（未修饰的成纤维细胞）加1ml培养基处理，III组仅接受1ml培养基。皮瓣缝合回原处，在接下来的6天内，每组10只动物双侧结扎并离断血管蒂。7天后，收获皮瓣，测量坏死量并进行组织学检查。
通过ELISA测定，GMFB产生的PDGF - AA比NMFB多高达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在，无明显炎症反应。在所有GMFB处理的皮瓣中，均可证明有大量血管生成。
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，产生大量具有血管生成活性的PDGF - AA。将这些细胞注射到腹直肌筋膜后，可诱导功能性血管生成，从而在该皮瓣模型中允许更早地离断血管蒂。
