Machens Hans-Günther, Morgan Jeffrey R, Berthiaume Francois, Stefanovich Peter, Siemers Frank, Krapohl Björn, Berger Alfred, Mailänder Peter
Clinic for Plastic, Hand and Reconstructive Surgery, Burn Center, Lübeck University Clinics, Ratzeburger Allee 160, 23562 Lübeck, Germany.
Surgery. 2002 Apr;131(4):393-400. doi: 10.1067/msy.2002.121887.
The aim of this study was to induce therapeutic angiogenesis in ischemically challenged flap tissue by means of gene transfer.
Isogenic rat fibroblasts were retrovirally transfected to produce platelet-derived growth factor (PDGF)-AA. Stable gene expression was monitored by PDGF-AA enzyme-linked immunosorbent assay. Eighty animals were divided into 2 groups (1 and 2), each with 4 subgroups. The angiogenic target was a 7 x 7-cm epigastric island flap used as a necrosis model. Group 1 received flap treatment 1 week before flap elevation: 10(7) genetically modified fibroblasts, expressing PDGF-AA (genetically modified fibroblasts) plus 1 mL of Dulbecco's modified Eagle's medium (DMEM) (1A), 10(7) nonmodified fibroblasts (NMFB) plus 1 mL of DMEM (1B), 1 mL of DMEM (1C), and 1 mL of sodium chloride 0.9% (1D). All substances were injected at evenly distributed spots into the panniculus carnosus of the entire flap. Group 2 had the same flap treatment at the day of flap elevation. All flaps were sutured back. Seven days later, the flaps were harvested and examined both clinically, histologically, and immunohistochemically.
In vitro, the GMFB produced up to 117.9 +/- 57.2 ng of PDGF-AA/mL medium during a 4-day period, compared with 0.7 +/- 0.6 ng of PDGF-AA/mL medium produced by NMFB in the same time period. In vivo production of PDGF-AA in flaps amounted to 1.3 +/- 0.7 ng of PDGF-AA/1 microL flap tissue for group 1A and 1.7 +/- 1.1 ng of PDGF-AA/1 microL flap tissue for group 2A seven days after cell transplantation. Fibroblasts persisted in all flaps from groups 1A, 1B, 2A, and 2B without major inflammatory reaction. Clinically, group 2A developed significantly less flap necrosis compared with all other groups, including group 1A. Accordingly, only group 2A gave significant histologic and immunohistochemical evidence for enhanced angiogenesis within the flap tissue.
After retroviral gene transfer, isogenic rat fibroblasts produce high amounts of PDGF-AA in vitro. In vivo, PDGF-AA can be detected in flaps receiving genetically modified fibroblasts, which suggests survival of the implanted fibroblasts in this model. PDGF-AA produced by GMFB can induce flap angiogenesis only under ischemic conditions in this model. Transplantation of PDGF-AA-overexpressing fibroblasts results in higher flap survival in this model.
本研究的目的是通过基因转移在缺血性皮瓣组织中诱导治疗性血管生成。
用逆转录病毒转染同基因大鼠成纤维细胞以产生血小板衍生生长因子（PDGF）-AA。通过PDGF-AA酶联免疫吸附测定法监测稳定的基因表达。80只动物分为2组（1组和2组），每组又分为4个亚组。血管生成的靶标是一个7×7厘米的腹壁岛状皮瓣，用作坏死模型。第1组在皮瓣掀起前1周接受皮瓣处理：10⁷个表达PDGF-AA的基因修饰成纤维细胞（基因修饰成纤维细胞）加1毫升杜氏改良伊格尔培养基（DMEM）（1A），10⁷个未修饰的成纤维细胞（NMFB）加1毫升DMEM（1B），1毫升DMEM（1C），以及1毫升0.9%氯化钠溶液（1D）。所有物质均注射到整个皮瓣的肉膜层中分布均匀的部位。第2组在皮瓣掀起当天进行相同的皮瓣处理。所有皮瓣均缝合回原处。7天后，切取皮瓣并进行临床、组织学和免疫组织化学检查。
在体外，基因修饰成纤维细胞在4天内每毫升培养基产生高达117.9±57.2纳克的PDGF-AA，而同期未修饰成纤维细胞每毫升培养基产生0.7±0.6纳克的PDGF-AA。细胞移植7天后，第1A组皮瓣中PDGF-AA的体内产量为1.3±0.7纳克/1微升皮瓣组织，第2A组为1.7±1.1纳克/1微升皮瓣组织。在第1A、1B、2A和2B组的所有皮瓣中，成纤维细胞持续存在，无明显炎症反应。临床上，与包括第1A组在内的所有其他组相比，第2A组皮瓣坏死明显较少。相应地，只有第2A组在皮瓣组织内有明显的组织学和免疫组织化学证据表明血管生成增强。
逆转录病毒基因转移后，同基因大鼠成纤维细胞在体外产生大量PDGF-AA。在体内，在接受基因修饰成纤维细胞的皮瓣中可检测到PDGF-AA，这表明在该模型中植入的成纤维细胞存活。在该模型中，基因修饰成纤维细胞产生的PDGF-AA仅在缺血条件下可诱导皮瓣血管生成。过表达PDGF-AA的成纤维细胞移植可使该模型中的皮瓣存活率更高。
