Mithani Suhail K, Bluebond-Langner Rachel, Rodriguez Eduardo D
R Adams Cowley Shock Trauma Center, University of Maryland School of Medicine and Johns Hopkins Medical Institutions, Baltimore, MD 21201, USA.
Ann Plast Surg. 2009 Sep;63(3):323-6. doi: 10.1097/SAP.0b013e318190320a.
Clinical detection of free flap failure lacks sensitivity. Failure is likely accompanied by altered gene expression; however, a genomic approach that identifies potential biomarkers and therapeutic targets has not been described. This study identifies genetic RNA expression alterations via microarray in a free flap failure animal model. A free tissue transfer rat model based on the inferior epigastric vessels was utilized. After microscopic anastomosis, the vein was occluded and RNA extracted from flap tissue of failure and control groups. Gene expression of 3 experimental and control group samples was assessed with the Affymetrix GeneChip Rat 230 v2.0 microarray. Quantitative reverse transcription polymerase chain reaction was performed on RNA of genes identified on an additional 6 experimental and 7 control group flaps. Eight hundred ninety of 28,000 genes had greater than 2-fold expression differences between experimental and controls. Student t test and 2-way analysis of variance filtering with equal variance identified 53 genes with statistically significant differences. Hierarchical clustering by gene ontology identified 4 genes with likely involvement in failure pathogenesis: RT1 class II, locus Bb, secreted frizzled-related protein 1, platelet/endothelial cell adhesion molecule, and Claudin 5. Validation performed by quantitative reverse transcription polymerase chain reaction revealed statistically significant expression alterations in locus Bb, platelet/endothelial cell adhesion molecule, and Claudin 5 of the failure group. Utilizing expression thresholds for test positivity, venous occlusion was predicted with 100% sensitivity and 86% specificity. Three highly sensitive and specific novel genes predictive of flap failure from venous occlusion were identified with altered expression in an animal model.
游离皮瓣失败的临床检测缺乏敏感性。失败可能伴随着基因表达的改变；然而，尚未描述一种能够识别潜在生物标志物和治疗靶点的基因组方法。本研究通过微阵列在游离皮瓣失败动物模型中鉴定基因RNA表达改变。使用了基于腹壁下血管的游离组织移植大鼠模型。显微吻合后，阻断静脉，并从失败组和对照组的皮瓣组织中提取RNA。用Affymetrix GeneChip Rat 230 v2.0微阵列评估3个实验组和对照组样本的基因表达。对另外6个实验组和7个对照组皮瓣中鉴定出的基因的RNA进行定量逆转录聚合酶链反应。28000个基因中有890个在实验组和对照组之间的表达差异大于2倍。采用学生t检验和等方差双向方差分析筛选出53个具有统计学显著差异的基因。通过基因本体进行层次聚类，确定了4个可能参与失败发病机制的基因：RT1 II类、位点Bb、分泌型卷曲相关蛋白1、血小板/内皮细胞黏附分子和Claudin 5。通过定量逆转录聚合酶链反应进行的验证显示，失败组的位点Bb、血小板/内皮细胞黏附分子和Claudin 5存在统计学显著的表达改变。利用检测阳性的表达阈值，预测静脉阻塞的敏感性为100%，特异性为86%。在动物模型中，通过表达改变鉴定出3个预测静脉阻塞导致皮瓣失败的高敏感性和特异性新基因。
