| BackgroundIn the end-to-end anastomosis of trachea, the resectable length is restricted to 1/2 of the total length (6 cm) in adults or 1/3 in children, and replacement of longer sections will only be feasible if a safe and functional tracheal replacement can be developed, such as tracheal allografts, artificial or tissue-engineered airway, autogenous tissues graft and so on. Among them, tracheal allografts may be the most effective option for achieving airway reconstruction at present. Although more and more researchers and clinicians focus on tracheal transplantations, there are a few clinical reports of tracheal allotransplantations. And immune rejection is one of key obstacles in tracheal allografts. Immunosuppressive therapy increases the possibility of sever infection and malignant tumor, while improving the allografts. Therefore it is necessary to find an alternative to suppress the immune rejection after tracheal transplantation.As is known, the immune rejection after allografts can be suppressed by eliminating the antigenicity of grafts. It was reported that tracheal antigenicity may be eliminated or reduced by the removal of epithelium in trachea. However, epithelial tissues play a key role in functional reconstruction and transplantation of trachea. Therefore, it is the best way to promote reepithelization from the recipient after the removal of donor epithelium.Heretofore, it is rare that the study of reepithelization from the recipient after the removal of donor epithelium in tracheal allografts. Thus the key steps in reepithelization process are not clear, the technique means are imperfect, and the theoretical basis is very poor. We plan to use enzymatic dissociation with trypsin to remove the epithelium from the rat donor trachea which is then colonized by epithelial cells that have been cultured from cells taken from the recipient. Afterwards, the donor trachea is heterotopically transplanted to the dorsum of recipient rat and reepithelization will be completed. And our objectives are to objectively evaluate the present method, to detailedly describe the whole process and to optimize the technique means.PART ONECulture and perfusion of epithelial cells from rat trachea, production of donor nude trachea and establishment of heterotopic tracheal transplantation modelObjectiveWe plan to perfuse the epithelial cells cultured from the recipient trachea into the donor nude trachea in which donor epithelial tissues has been removed by enzymatic dissociation in order to reduce the antigenicity of donor trachea and complete reepithelization from the recipient. It is the key steps that culture and perfusion of epithelial cells from rat trachea, production of donor nude trachea and establishment of heterotopic tracheal transplantation model.MethodsThe tracheal epithelial cells of SD rats were primarily cultured in Ham's F-12 and the cultured cells were diluted to cell suspension (10×106 cells/ml).Wistar rats (n=80) were taken as donors and SD rats (n=80) as recipients. Tracheas (1.5-2.0 cm) were excised from below the larynx to the major bronchi. The explanted tracheas were digested for 18 h with 0.25% trypsin at 4℃, then the qualification of donor nude tracheas were evaluated by HE stain. The qualified donor nude tracheas were stored in Ham's F-12. One end of donor nude trachea was ligated, and the recipient cell suspension was perfused into its lumen before ligation of another end. Afterwords, donor tracheas were heterotopically subcutaneous transplanted to the dorsum of recipients.ResultsEpithelial cells being round-shape have good diopter and suspended in culture solution. After 24 hours, the majority of cells started to adhere to the wall. After 3 days, cells showed typical "flagstone" appearance. After 4-5 days, cells formed the flake-shape cell clusters. HE stain showed that epithelial cells were totally removed from the donor trachea with integrated basement membrane and normal cartilage tissues. The mean time of animal model establishment is 5.2±1.5 min. There was no postoperative complications, eg. infection, and the survival rate was 100%.ConclusionThe enzymatic dissociation with trypsin is a safe and effective method to remove epithelial cells from the donor trachea. Ham's F-12 is a suitable culture solution for our study. Perfusion of cells suspension is a simple and available technique to complete the epithelial implantation. Because of easy operation and high success rate, heterotopic subcutaneous tracheal transplantation model in rat is the best choice in present study.PART TWOEffect of immunosuppressive therapy on reepithelization after rat heterotopic tracheal graft with recipient epithelial cells perfusionObjectiveAfter totally removal of epithelial cells, there are still MHC antigens existing in rest cells and tissues, which means donor nude trachea still has antigenicity and immune rejection will take place after transplantation. And we need to answer the following questions:Will immune rejection has adverse influence on reepithelization from the recipient? Is it necessary to perform the immunosuppressive therapy? What is the best treatment regimen of immunosuppressive therapy? How is reepithelization after rat heterotopic tracheal graft with recipient epithelial cells prefusion?MethodsAfter establishment of heterotopic subcutaneous tracheal transplantation model in rat, animals were randomly divided into six groups. Control group 1 (n=10):normal saline was perfused into donor nude trachea, without immunosuppressive therapy; Control group 2 (n=10):recipient epithelial cells suspension was perfused into donor nude trachea, without immunosuppressive therapy; Test group 1 (n=10):recipient epithelial cells suspension was perfused into donor nude trachea, with intramuscular injection of Tacrolimus (0.5 mg/kg/d) only for three consecutive days after transplantation; Test group 2 (n=10):recipient epithelial cells suspension was perfused into donor nude trachea, with intramuscular injection of Tacrolimus (0.5 mg/kg/d) for seven consecutive days after transplantation; Test group 3 (n=10):recipient epithelial cells suspension was perfused into donor nude trachea, with intramuscular injection of Tacrolimus (1.0 mg/kg/d) only for three consecutive days after transplantation; Test group 4 (n=10):recipient epithelial cells suspension was perfused into donor nude trachea, with intramuscular injection of Tacrolimus (1.0 mg/kg/d) for seven consecutive days after transplantation. All rats were killed on 28 days after transplantation and then the implanted tracheas were harvested. In all specimens, histological and ultrastructural changes were evaluated, infiltrated lymphocytes were counted, differentiation of epithelium (CK14, CK18, CFTR) were identified, percentage of cilia and fibrosis tissues were calculated.ResultsGenerally the results of test groups were better than control groups. In the latter, there was not any epithelial regeneration, and tracheal stenosis, even lumen occlusion, were found. Test group 1 and test group 2 had the similar results that immature epithelial regeneration took place in the regional area of tracheal inner surface. And test group 3 and test group 4 also had the similar results that mature ciliated columnar epithelium covered the whole inner surface of trachea.ConclusionIt is an effective way to complete reepithelization from the recipient after rat heterotopic tracheal graft with recipient epithelial cells injection. And short-term and low dosage immunosuppressive therapy is necessary. The best regimen is the intramuscular injection of Tacrolimus (1.0 mg/kg/d) for three consecutive days after transplantation. Moreover, epithelial cells from the recipient may inhabit the development of obliterated airway disease.PART THREEStudy on the process of reepithelization after rat heterotopic tracheal graft with recipient epithelial cells perfusionObjectiveIn part two, we have improved that it is an effective way to complete reepithelization from the recipient that heterotopic tracheal graft with recipient epithelial cells perfusion in rat with short-term and low dosage immunosuppressive therapy. But it is still unclear about the detailed process of reepithelization.MethodsAfter establishment of heterotopic subcutaneous tracheal transplantation model in rat, the animals were randomly divided into six groups. All animals were intramuscular injected of Tacrolimus (1.0 mg/kg/d) only for three consecutive days after transplantation. Rats (n=10) were killed on 3,7,14,21,28 and 35 days respectively after transplantation and then the implanted tracheae were harvested. In all specimens, histological and ultrastructural changes were evaluated, differentiation (CK14, CK18, CFTR) and proliferation (Ki67) of epithelium were identified, percentage of cilia were calculated.ResultsOn 3 days after transplantation, the regional areas of tracheal inner surface were covered by non-ciliated flate cells with normal cellular ultrastructure and wide intercellular space, but without cell junctions. And the levels of CK14, CK18, CFTR and percentage of cilia were low, while Ki67 was high.On 7 days, the whole areas of tracheal inner surface were covered by non-ciliated flate cells. And the rest results were similar to those on 3 days after transplantation.On 14 days, the whole areas of tracheal inner surface were covered by the mixture of flate cells and columnar cells with sparse cilia. The latter arranged in pseudostratified structure which consisted of goblet cells, basal cells and so on. Cell junctions were found under Transmission Electron Microscope. The levels of CK14, CFTR were still low, but the level of CK18 and percentage of cilia started to increase and the level of Ki67 started to decrease.On 21 days, the whole areas of tracheal inner surface were covered by columnar cells. The majority of cells arranged in pseudostratified structure. The level of CK14 was still low, and percentage of cilia still increased and the level of Ki67 decreased. But the level of CFTR started to increase and CK18 reached the peak level.On 28 days, the whole areas of tracheal inner surface were covered by ciliated columnar cells arranged in pseudostratified structure. The narrow intercellular space and tight cell junctions were observed under Transmission Electron Microscope. The levels of CK14, Ki67 were low, and the levels of CK18, CFTR and percentage of cilia were high.The results on 35 days after transplantation were similar to those on 28 days.ConclusionCells adhered to tracheal inner wall quickly after perfusion of cells suspension. Then the adherent flate cells started to proliferate until the whole inner surface was covered. And the cells started to differentiate. Finally, mature pseudostratified ciliated columnar cells appeared.
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