| BackgroundThe number of patients requiring tracheal reconstruction has been rising since the increase of morbility rate of iatrogenic tracheal stenosis, complex tracheal injury, primary and secondary tracheal tumors in recent years. None of the current methods for tracheal reconstruction is perfect while tracheal allotransplantation is relatively acceptable since the reconstruction can be conducted both structurally and functionally, which makes it one of the most promising approaches for clinical application. Tracheal cicatricial stenosis caused by OAD(Obliterative Airway Disease), however, is one of the main postoperative complications after tracheal allotransplantation and the primary cause of the failure of tracheal transplantation. EMT(Epithelial to Mesenchymal Transition) is that normal epithelial cells lose their epithelial characteristics and obtain mesenchymal phenotypes. Meanwhile, the expression of epithelial markers decreases whereas the expression of mesenchymal markers and EMT-related transcriptional factors increases. According to previous reports, EMT exists widely in histoembryological development, invasion and metastasis of malignancies, and tissue fibrosis. One of the typical characteristics after tracheal allotrasplantation is that there is massive fibrotic connective tissue inside the narrowed graft lumen. In addition, it has been reported that human primary bronchial epithelial cells can undergo EMT after the administration of TGF-β1(Transforming Growth Factor-β1). Therefore, we infer that EMT might be one of the important factors that induce allograft stenosis. Another study shows that the coverage rate of tracheal epithelia is negtively correlated to the extent of airway occlusion, which means the lower the coverage rate of tracheal epithelia is, the more severe the extent of tracheal occlusion will be. It can be concluded that the injury of donor tracheal epithelial cells will eventually lead to graft stenosis. However, it is still not clear whether the injury causes graft stenosis by inducing EMT, and it is poorly understood whether seeding of recipient-originated epithelial cells can attenuate graft stenosis via inhibiting EMT. ObjectiveTo investigate the role of tracheal epithelial cell in tracheal allograft stenosis, and elucidate the possible molecular and biological mechanism for how seeding of tracheal epithelial cells inhibits tracheal allograft stenosis, clinically providing new insights into the precaution and theraputics of tracheal stenosis after allotransplantation. Methods(1) Wistar rat primary tracheal epithelial cells were isolated and cultured by Accutase enzymatic digestion.(2) 3 groups of rat dorsal subcutaneous heterotopic transplantation model were established, including SD-Wistar rat allotransplantation model, SD-SD rat homotransplantation model and SD-Wistar rat allotransplantation model with seeded recipient-originated epithelial cells(Wistar-originated epithelial cells were seeded 6 days after transplantation). The subject animals were sacrificed at days 7, 14 and 30, and the tracheal grafts were collected for paraffin embedding, total RNA and protein extraction. Paraffin sections were prepared for H&E(Hematoxylin-Eosin) staining and a pathological image analysis system was utilized to exam tracheal epithelial cell coverage rate, inflammatory infiltration and lumenal occlusion.(3) Paraffin sections were prepared for IHC(Immunohistochemistry) to test the location and expression of E-cadherin(epithelial marker) and vimentin(mesenchymal marker). Then total protein was extracted for Western Blot to further determine the expression of E-cadherin and vimentin. Total RNA was extracted and afterwards reversely transcripted into c DNA for q RT-PCR(Quantitative Real-Time Polymerase Chain Reaction) to test the expression levels of ZEB1, ZEB2, Snail1, Snail2 and Twist1. Western Blot was applied to examine the protein levels of ZEB1.(4) TGF-β signaling related molecules were determined, including TGF-β1, TGF-β2, TGF-β3 and TGFBR1(Transforming Growth Factor-β Receptor 1). Smad3 and p-smad3 were tested by Western Blot. Results(1) The optimum isolating conditions of rat tracheal epithelial cell isolation was explored by Accutase enzymatic digestion, and Wistar rat tracheal epithelial cells were isolated successfully.(2) Rat dorsal subcutaneous heterotopic transplantation model was successfully established. The grafts were then subjected to histopathological examination. Syngrafts retained basically normal histopathological structure, while the number of tracheal ciliated epithelial cells decreased gradually and the extent of lumenal occlusion and inflammatory infiltration aggravated progressively in allografts. The number of tracheal ciliated epithelial cells restored in seeded allografts, and the extent of lumenal occlusion and inflammatory infiltration was attenuated.(3) IHC revealed that E-cadherin was mainly expressed in epithelial cells while vimentin in mesenchymal cells. The expression levels of E-cadherin were high in syngrafts while decreased in allografts. The expression of E-cadherin increased after epithelial cell seeding. On the contrary, the expression pattern of vimentin was just the opposite of E-cadherin. And the expression of E-cadherin and vimentin was further validated by Western Blot.(4) EMT related transcriptional factors were tested by q RT-PCR. Compared to isografts, the expression levels of EMT related transcriptional factors were all up-regulated, of which the increase of ZEB1 was most obvious. The expression levels of EMT related transcriptional factors were dampened, and the inhibition on expression of ZEB1 was most evident in seeded allografts. The conclusion was further verified by Western Blot.(5) The expression levels of TGF-β1, TGF-β2, TGF-β3 and TGFBR1 were all up-regulated in allografts relative to syngrafts while the up-regulation of TGF-β1 expression was most obvious. Meanwhile, the phosphorylated level of smad3 was elevated in allografts compared to syngrafts. In seeded allogeneic group, the expression levels of TGF-β1 and TGFBR1 were down-regulated, as well as the phosphorylated level of smad3. Conclusions(1) Donor tracheal epithelial cell injury and lumenal occlussion occur after rat heterotopic tracheal allotransplantation.(2) Recipient-orginated epithelial cell seeding ameliorates tracheal stenosis in rat heterotopic tracheal allotransplantation.(3) The activation of TGF-β signaling cascade by tracheal epithelial cell injury is one of the pivotal molecular mechanisms that motivate EMT in tracheal epithelial cells and aggravate tracheal stenosis.(4) Seeding of recipient tracheal epithelial cells prevents the progression of OAD by attenuating EMT via TGF-β-Smad signaling in rat heterotopic tracheal allotransplantation. |
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