腺病毒介导外源基因经动脉转染大鼠腹壁浅动脉岛状皮瓣的研究

Adenovirus mediated exgenous gene transfect rat's SIEA flap by intra-artery perfusion

目的:探讨腺病毒载体介导外源基因经动脉灌注转移修饰大鼠腹壁浅动脉岛状皮瓣(Superficial inferior epigastric artery flap,SIEA)的可行性、安全性并对其转染条件作出评价。方法:本研究通过血管夹暂时夹闭形成局部封闭环境,应用腺病毒介导增强型绿色荧光蛋白基因(Ad5-CMV-EGFP)经动脉灌注带蒂SIEA60min。60只SD大鼠随机分为6组:A、B、C、D组分别为:3.0×106、3.0×107、3.0×108、3.0×109pfu/ml,E组:PBS、F组:腺病毒空载体。术后7天应用活体荧光成像系统观察皮瓣的荧光强度,应用共聚焦显微镜、RT-PCR检测皮瓣组织中EGFP的表达和分布情况,通过组织学分析对皮瓣的安全性影响。结果:术后7天B组、C组、D组活体观察皮瓣可见体表绿色荧光信号。共聚焦显微镜观察结果与活体观察结果一致,EGFP在皮瓣内血管内、外膜均可见表达,血管周围组织包括脂肪、毛囊可见EGFP表达,皮瓣外组织未见EGFP表达。对照组E组、F组未见EGFP表达。RT-PCR结果显示,EGFP仅在皮瓣内表达,其他重要脏器(心脏、肝脏、肾脏)无表达。对皮瓣组织学观察未见明显炎症反应。结论:复合组织瓣转移修复创面是一种成熟的手术方式。本研究从大体观察、组织形态学和分子生物学三方面评价外源基因经动脉内灌注转染大鼠带蒂皮瓣的可行性,为进一步深入研究基因修饰复合组织瓣。

Objective To study the feasibility of superficial inferior epigastric artery （SIEA） flap transfected by intra-artery perfusion with exogenous gene mediated by adenovirus, and assess the transfect efficiency and optimal conditions. Methods： In our study ,rat＇s SIEA flap is transfected by intra-artery perfusion with exogenous enhanced gene-green florescent protein mediated by adenovirus （Ad5-CMV-EGFP） for 60 min . 60 Sprague Dawley rats were randomly divided into 6 groups： A （3.0×10^6pfu/ml）, B（3.0×10^7pfu/ml）, C （3.0×10^8pfu/ml）, D（3.0×10^9pfu/ml）, E（PBS）, F（Adenovirus）, and assess the transfect efficiency and optimal conditions. Under the optimal conditions, various components of SIEA flaps and the expressions of exogenous genes in adjacent tissue are observed to evaluate the effects of this transfect model to the whole system. At the 7th day of post operation, EGFP expression in tissue was detected by IVIS Imaging System, laser scanning confocal microscope and RT-PCR .Histological evaluation was used to assess toxicity. Results： The expression of EGFP was detected by IVlS Imaging System in group B, group C, group D at the 7th day of post operation. The result detected by fluorescence microscopy was similar to IVIS Imaging System. Ad5-CMV-EGFP.EGFP expression was comfirmed in the flap＇s intima, and perivascular tissue including adipose tissue, follicles. The result of RT-PCR shows that the sequence of EGFP is observed in flap, but no EGFP was observed in heart,liver or kidney. Histological evaluation of the flap revealed no evidence of inflammation in the treated group. Conclusion Wound reconstruction by using microvascular flaps is a mature measure in plastic surgery. In this research, the author study the feasibility to transfect exogenous genes to rat＇s pedicle by intra-artery perfusion. By doing this, we can further study the genetically modified microvascular pedicle flaps ,and provide theoretic references for applying pedicle composite tissue flap to perform therapeutic and reconstructive functions.