The Inhibitory Effect Of Transfection Of TRADD Gene-expressing Lentiviruses Using Stents As Carriers In Benign Esophageal Stricture

Research backgroundDysphagia caused by benign esophageal stricture is difficult to handle in clinical practice; it severely affects patients’ quality of life and can induce serious complications. Its treatment usually includes bouginage or balloon dilation; however, these techniques have high recurrence rates. Currently, the treatment for refractory benign esophageal stricture usually uses esophageal stent placement. Esophageal stents can significantly improve symptoms such as dysphagia; however, these stents still have some limitations. The metal components of both ends of self-expandable metal stents(SEMS) have stimulation effects on esophageal mucosa and usually cause granulation tissue hyperplasia to induce re-stricture; in addition, granulation tissues usually embed stents, which can cause esophageal rupture upon removal of metal stents. Self-expandable plastic stents(SEPS) can reduce reactive tissue hyperplasia and can increase stent removal safety; however, SEPS have higher stent migration rates( > 62%) and shorter dysphagia remission time. Biodegradable stents(BDS) allow stents to be embedded by tissues and do not require removal; however, because BDS only have a 6-week lifetime, they cannot effectively support for more than 8-12 weeks and thus have high re-stricture rates. Therefore, currently, there is no satisfactory treatment measure for refractory benign esophageal stricture.Benign esophageal stricture is mainly caused by hypertrophic scars. Current studies have indicated that apoptosis is closely associated with scar hyperplasia and keloid formation. The presentation of apoptosis is gene expression along apoptosis signaling pathways. The major transduction pathway is death receptor(DR)-mediated signal transduction. Current studied death receptors include tumor necrosis factor receptor 1(TNFR1), FAS, DR3, DR4, DR5, DR6, nerve growth factor receptor(NGFR), and ectodysplasin A receptor(EDAR). Among them, TNFR1 is the most thoroughly studied. TNFR1 interacts with its ligand, tumor necrosis factor receptor-associated death domain protein(TRADD), to regulate cell proliferation and apoptosis through the tumor necrosis factor alpha(TNF-α)-mediated signaling pathways. In scar tissues, the low level of TRADD expression might inhibit TNF-α-mediated apoptosis pathways, thus resulting in excessive proliferation of hypertrophic scar fibroblasts(HSFbs) and pathological scar formation. Our previous study successfully constructed human TRADD gene-overexpressing lentiviruses using the lentiviral vector system. After transfection into HSFbs, the recombina nt viruses could restore TRADD protein expression in HSFbs, inhibit HSFb cell proliferation, influence cell division, promote cell apoptosis, and reduce collagen I protein secretion, thus reducing HS formation.Therefore, we aimed to use argon plasma coagulation(APC) to construct an esophageal stricture model in experimental dogs and to use fully covered esophageal stents to transfer TRADD gene-expressing lentiviruses into tissues at esophageal stricture segments to observe the effects on tissue hyperplasia inhibition and re-stricture prevention.ObjectiveThis study observed the effect of TRADD gene transfection in the mucosa of benign esophageal stricture in dogs and its inhibitory effect on esophageal stricture after esophageal stents coated with TRADD gene-expressing lentiviruses were installedMethods1.Fifteen experimental dogs were randomly and evenly divided into experimental, control, and blank groups. Benign esophageal stricture models were constructed using argon plasma coagulation(APC). Covered esophageal stents coated with GFP-TRADD gene-expressing lentiviruses, esophageal stents coated with green fluorescence protein(GFP)-expressing lentiviruses, and regular covered esophageal stents were placed into dogs, and the conditions of stent loss were observed after one week. Stents were removed, and the degrees of stricture were observed using gastroscopy after 2, 3, and 4 weeks.2.Experimental dogs were sacrificed after 4 weeks, and pathological and immunofluorescence examinations were performed on esophageal stricture tissues.3.Western blotting was performed to detect TRADD, collagenⅠ, collagenⅢ, and α-smooth muscle actin(α-SMA) protein expression in esophageal tissues in the three groups.1.One week after esophageal stent placement, gastroscopy results showed that all covered stents in dogs in the experimental group had migrated into the stomach, while covered stents migrated into the stomach in only 2 and 3 dogs in the control and blank groups, respectively. Non-migrated esophageal stents in experimental dogs were removed by gastroscopy. After removal, the diameters of esophageal stricture sites were measured every week. Significant esophageal stricture occurred again in experiment dogs in the control and blank groups, while the re-stricture in the experimental group was milder. Repeated measures designed multivariate analysis of variance was performed, and P<0.01 indicated statistical significance.2.Hematoxylin and eosin(HE) staining results of esophageal stricture tissues indicated that hyperplasia of granulation tissues and fibrous tissues occurred in the blank and control groups, while hyperplasia of granulation tissues and fibrous tissues was not obvious in the experimental group. Immunofluorescence microscopy revealed GFP expression i n submucosal tissues, indicating that TRADD gene-expressing lentiviruses and lentiviruses grew in esophageal submucosa.3.Western blot detection results showed that the GFP-TRADD-Flag protein could be 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 collagenⅠ, collagenⅢ, and α-SMA protein expression levels in the experimental group we re significantly lower than those in the other two groups; the differences were statistically significant.ConclusionUsing stents as carriers resulted in successful transfer of TRADD-expressing lentiviruses into esophageal mucosal tissues, which could inhibit tissue hyperplasia at stricture sites and could decrease re-stricture of esophageal stricture sites.