以支架为载体的TRADD基因慢病毒转染对食管良性狭窄的抑制作用

Inhibitory effect of TRADD gene-expressing lentivirus vector transfection with covered stent as carrier in canine benign esophageal stricture

目的观察TRADD基因慢病毒涂层食管支架置入后,TRADD基因对犬食管良性狭窄黏膜的转染效果及对食管狭窄的抑制作用。方法采用随机数字表法,将15只实验犬随机平均分为实验组、对照组及空白组,采用氩离子凝固器制作良性食管狭窄模型,分别置入GFP-TRADD基因慢病毒涂层带膜食管支架、GFP-慢病毒涂层食管支架、普通带膜食管支架,1周后观察支架脱落情况,并取出支架,2、3、4周胃镜下观察狭窄程度。4周后处死实验犬,对狭窄食管组织行病理、Masson三色染色法、免疫荧光检查,分别采用Western blot检测TRADD蛋白在3组食管组织中的表达,同时检测collagenⅠ、collagenⅢ、α-SMA 3种蛋白在食管组织中表达情况。结果置入食管支架1周后,胃镜检查见实验组实验犬带膜支架全部移位进入胃,对照组和空白组实验犬分别有2、3只实验犬其带膜支架移位进入胃中,胃镜取出所有实验犬未移位食管支架。此后每周测量其食管狭窄处直径,可见对照组及空白组实验犬再次出现食管明显狭窄,而实验组再狭窄较轻。采用重复测量设计的多因素方差分析具有统计学差异(P<0.01)。食管狭窄组织HE染色结果表明空白组及对照组肉芽组织及纤维组织增生,而实验组肉芽组织及纤维组织增生较不明显。Masson染色结果表明实验组胶原纤维较空白组、对照组明显减少(P<0.05)。免疫荧光镜检查可见绿色荧光蛋白在黏膜下组织,表明TRADD基因慢病毒及慢病毒均在食管黏膜下生长。Western blot检测实验组可检测GFP-Tradd-Flag蛋白,而空白组和对照组未检测出该蛋白。Western blot检测显示实验组collagenⅠ、collagenⅢ、α-SMA 3种蛋白较其他两组明显降低(P<0.05)。结论以支架作为载体可成功将TRADD慢病毒转入食管黏膜组织中,可抑制狭窄部位组织增生,减少食管狭窄部位再狭窄发生。

Objective To determine the effect of transfection of lentivirus vectors carrying tumor necrosis factor receptor associated death domain（ TRADD） gene in the canine mucosa of benign esophageal stricture esophageal stents coated with TRADD gene-expressing lentivirus viruses on esophageal stricture after implanting. Methods Fifteen experimental dogs were evenly divided into experimental,control,and blank groups（ n = 5）. Benign esophageal stricture models were constructed using argon plasma coagulation（ APC）.Covered esophageal stents coated with GFP-TRADD gene-expressing lentivirus vector,esophageal stents coated with green fluorescence protein（ GFP）-expressing lentiviruses,and regular covered esophageal stents were placed into the dogs from the 3 groups respectively,and the conditions of stent loss were observed after1 week. Stents were removed,and the degrees of stricture were observed using gastroscopy in 2,3,and4 weeks after implantation. Experimental dogs were sacrificed after 4 weeks,and Masson Trichrome staining and immunofluorescence assay were performed on esophageal stricture tissues for pathological changes.Western blotting was performed to detect the protein expression levels of TRADD,collagen Ⅰ,collagen Ⅲ,and α-smooth muscle actin（ α-SMA） in esophageal tissues in the 3 groups. Results In 1 week after esophageal stent placement,gastroscopy results showed that all covered stents in the dogs had migrated into the stomach in the experimental group,while the covered stents migrated into the stomach in only 2 and 3 dogs in the control and blank groups,respectively,and those non-migrated esophageal stents were removed by gastroscopy. After removal,the diameters of esophageal stricture sites were measured every week. Significant esophageal stricture occurred again in the dogs of the control and blank groups,while the re-stricture in the experimental group was much milder（ P 〈 0. 01） by repeated measures designed multivariate analysis.Hematoxylin and eosin（ HE） staining indicated that the hyperplasia of granulation tissues and fibrous tissues was found in the esophageal stricture tissues of the blank and control groups,while no such obvious changes were seen in the experimental group. Masson Trichrome staining revealed that there were significantly less collagen fibers in the experimental group than the blank group and control group（ P 〈 0. 01）.Immunofluorescence assay displayed that GFP was expressed in the submucosal tissues, indicating that TRADD gene-expressing lentiviruses grew in the esophageal submucosa. Western blotting showed that the GFP-TRADD-Flag protein was detected in the experimental group,while the protein was not detected in the blank and control groups,indicating that TRADD gene-expressing lentiviruses were successfully transfected into esophageal submucosal tissues. Western blot results showed that the expression levels of collagen Ⅰ,collagenⅢ,and α-SMA protein were significantly lower in the experimental group than those in the other2 groups（ P 〈 0. 05）. Conclusion Using stents as carriers results in successful transfer of TRADDexpressing lentiviruses into esophageal mucosal tissues,which can inhibit tissue hyperplasia at stricture sites and decrease re-stricture of esophageal stricture sites.