动物造模,脱细胞异体气管移植 z-bio 2021-05-28 320

　　Method: Using a decontamination enzyme method (including sodium deoxycholate/deoxyribonucleic acid eluate), a semi-allotransplantation was taken from adult New Zealand white rabbits, and multiple elutions were performed as required (group 1, n = 7). The other group was kept in phosphate buffered saline at 4°C as a control (group 2, n = 7). One and two groups of trachea were transplanted into 14 age-matched rabbits. Results: In group 1 (decellularization), all rabbits survived, while in group 2 (control group), all rabbits died of airway obstruction 20 and 45 days after surgery. Histologically, the acellular allograft showed complete regeneration of epithelium and cartilage, while the fresh graft showed inflammatory changes without epithelial cells and cartilage. Conclusion: After acellular tracheal transplantation without immunity, the epithelium and cartilage ring are completely regenerated. The results showed that the decellularization process reduced the allogeneic tracheal response. Therefore, we believe that acellular allogeneic tracheal transplantation is a good way to replace the trachea. As far as we know, this is the first long-term (1 year) prognostic experiment to observe the transplanted trachea.

　　Keywords: Cell allogeneic rabbit tracheal transplantation

　　Introduction: The initial reconstruction of tracheectomy is the only effective way to treat various benign and malignant tracheal diseases. Unfortunately, the resectable length of the trachea in children is usually limited to 30% of the total length of children and about 6 cm in adults. The further increase in resection depends on the development of alternative methods of safe trachea. Almost all attempts to provide safe and reproducible autologous or artificial tracheal transplantation have been disappointing and questioned the future of long-term tracheal replacement, so to date, there are no clinically available transplants. Tissue bioengineering has become the most promising technology to create a near-normal trachea. To determine whether the decellularized trachea is a viable alternative to the replacement procedure, we evaluated the survival rate and histological changes of the decellularized tracheal transplantation in a rabbit transplant model without immunosuppression. Materials and methods: The allograft was taken from an adult male rabbit in New Zealand, weighing 3170-4575 g. The animals were anesthetized by intravenous injection of pentobarbital (30 mg/kg). Natural ventilation in the supine position allows harvesting or transplantation without tracheal intubation. The cervical trachea is exposed through a median neck incision and then removed from the cricoid cartilage to the nasal cavity. Collect grafts from beating heart donors without warm ischemia to obtain maximum graft survival. The harvested works are divided into two groups. One half (control) was placed in a stock solution made of phosphate buffered saline (PBS) containing 1% antibiotic and antifungal solution. Use the same method to store the other half for 24 hours and follow the steps below. The collected tracheal covering tissue was stripped, the tracheal muscles were stripped, and washed with a PBS solution containing 1% antibiotics and antifungal agents 4 times (4 hours each time). The tissue is placed in multiple treatment cycles, including the following steps: the tissue is stored in AQA at 4°C for 48 hours, and then incubated with 4% sodium deoxycholate for 4 hours. Incubate for 4 hours in NaCl containing 2000 kUDnase-I. Histology confirmed the presence of cytokines and major histocompatibility complex (MHC) cells. The bioengineering process lasts for 35 days, which is equivalent to 17 detergent-enzyme (DEM) cycles. Then, the bioengineered tracheal matrix showed fewer cartilage cells and fewer residual nuclei, but completely eliminated MHC I and II antigens. In contrast, MHCI and II are common in controlling the trachea. Rabbits whose transplantation age matches the age of the recipient. Under anesthesia, the recipient is placed in a supine position, and a 6-ring tracheal segment is removed through a median neck incision. Under anesthesia, the recipient is placed in a supine position, and a 6-ring tracheal segment is removed through a median neck incision. Allogeneic transplantation (control group, n=7; decellularization, n=7) was performed in an end-to-end, continuous manner using absorbent threads. The wound is closed in the usual way. Observe all 14 rabbits for 3 hours and then return them to the cage. Accept standard feed and water. On the first day after surgery, analgesia (buprenorphine, 0.05 mg/kg) was given twice a day. Enrofloxacin was taken at a dose of 10 mg/kg for 5 consecutive days after the operation. No immunosuppressants or steroids. Five days, fifteen days and three months after surgery, soft bronchoscopy was performed. From the 20th day to the 365th day, a pathological examination of the graft was performed.

　　Results: Summarize the results of 14 transplant recipients. In the decellularized group (group 1), the rabbit survived the experiment and was subsequently sacrificed for histological examination. In the control group (group 2), 7 rabbits died of airway obstruction 20, 25, 29, 34, 39, 41, and 45 days after surgery. Bronchoscopy showed signs of epithelial regeneration in and around the wall after transplantation, the airway was open, and the first group did not show stenosis or softening. However, in the second group, the airways were almost completely blocked by fibrosis and granulation tissue. Histologically, obvious lymphocyte and monocyte infiltration, severe fibrosis and cartilage ring destruction were observed in the two groups. In the first group, complete epithelial regeneration (well-differentiated respiratory epithelium) and cartilage tracheal ring regeneration were observed. No infiltration of lymphocytes and monocytes was observed in the first group.

　　Discussion: Compared with other successful organ replacements that occur in a sterile interstitial environment (liver, kidney, heart, etc.), the airway represents the interface between the mammal and the external environment. Mucosa plays the role of immunocompetent cells in airway transplantation. These can lead to acute transplant rejection and require large doses of immunosuppression. Various prostheses have been tested, but due to lack of epithelium, bacterial infection, excessive granulation due to obstruction, and anastomotic leakage, clinically suitable prostheses have not been developed. The reconstruction of various autologous tissues (such as the pericardium, periosteum, esophagus, and small intestine) can be reinforced with stents. Antigenicity studies have shown that human airway epithelium can form HLA-DR antigens, activate T lymphocytes, and play an important role in transplant rejection. The use of immunosuppressive agents to control the immune response can reliably suppress transplant rejection, but it can also increase respiratory infections after tracheal replacement. Therefore, reducing the antigenicity of the allograft itself is a good control method. For this reason, cryopreservation, radiotherapy and photodynamic therapy have been reported. The current results show that the DEM process requires a tracheal matrix of MHC antigens in living tissues while maintaining structural integrity similar to natural gas pipelines. Most importantly, this is a simple and effective method sufficient for potential clinical applications. length. .. In experiments, complete regeneration of epithelium and cartilage was observed after cell-free tracheal transplantation. There is no lymphocyte infiltration in or under the epithelial layer. During the bronchoscopy, the tracheal lumen was opened and no soft tissue changes were seen in the transplanted area. Experimental results show that acellular tracheal transplantation has good long-term tissue viability and reduced antigenicity.

　　Conclusion: After acellular tracheal transplantation without immunity, the epithelium and cartilage ring are completely regenerated. The decellularization process reduces the allogeneic response to the trachea.