Cryopreservation Of The Tracheal Antigen Presenting Cells And Tissue Activity Of Trachea

Objective: Due to congenital diseases, inflammation, cancer and other causes extensive tracheal stenosis in clinical practice is very common, therefore, need governor segment tracheal resection and reconstruction. Tracheal reconstruction of thoracic surgery at home and abroad is currently one of the thematic focus of the study, the trachea because of its unique physiological characteristics of anatomy, when its removal when the length of more than 6cm can not be fit because of tension too great. In order to maintain the airway smooth airway remodeling in a variety of methods have emerged. There are artificial tracheal replacement with autologous tissue on behalf of the trachea, bio-medicine and tissue engineering reconstruction of trachea and tracheal transplantation. Which tracheal transplantation is considered the preferred method of tracheal reconstruction. Tracheal transplant and other organs due to be as immunogenic after transplantation occur immune rejection, for the need for tracheal transplant patients, the majority of patients with adenocarcinoma and squamous cell carcinoma, the application immunosuppressive agents not only affect the postoperative recovery, and can lead to cancer recurrence, which in recent years, cryopreservation of the trachea after transplants were well received. refrigeration, including low-temperature (higher than -80℃), deep hypothermia (-80℃), ultra-deep low-temperature (-196℃), as well as methods such as repeated freezing and thawing. Frozen after the organization can maintain its original activity and tissue integrity, and can reduce their immunogenicity, then it will solve the preservation of the donor, but also solve the post-transplant immune rejection control problem.To understand the ultra-deep low-temperature (-196℃) under the conditions of tracheal tissue cell immunogenicity and viability, we used immunohistochemistry to observe the rat tracheal organizations morphological changes of dendritic cells, using 3H-TdR (3H-thymidine) method of in vitro cell viability.Methods: 48SD rats weighing 200-250g, purchased from the Animal Experimental Center, Hebei Medical University. Were randomly divided into six groups, one group will do the control group. 2.5-3.5cm were cut trachea, whichever is cutting edge, using immunohistochemical staining (OX-62 markers) to detect the expression of dendritic cells will be moved to fill the trachea, respectively cryoprotectant in frozen storage tubes (2ml), each tube put in a trachea, so that the full infiltration of cryoprotectant trachea, tighten tube cap. Placed on the balance between 4℃refrigerator 4 hours, using programmed cooling instrument procedures for cooling, cooling rate of 1℃/ min, down to -80℃to rapidly put in liquid nitrogen preservation. 24 hours, the first 10 days, 20 days, 30 days, 60 days, respectively, whichever is cut out 8 tracheal cartilage ring the edge of 4-6 months, after after rewarming compared with fresh trachea, the remaining trachea in vitro analysis of cell culture methods function and vitality, and to detect the expression of dendritic cells.Histopathology using HE staining of paraffin sections; immunohistochemistry with SP method. Application SPSS13.0 statistical software analysis test results.Results:1 Histological examination ultra-deep trachea after cryopreservation and after thawing compared with fresh trachea was no significant difference in appearance, slightly pale. The structure of the normal trachea: HE staining showed the trachea John membrane surface rows of pseudostratified ciliated columnar epithelial cells, mucosa and submucosa of stromal cells, glandular cells and vascular endothelial cells intact. Cartilaginous part of the compact structure, we can see the growth of cartilage cells, either alone or more within a lacuna in the cartilage. Frozen tracheal structure: the basic structure of each group to maintain integrity of the trachea, with time off portion ciliated, epithelial damage, cartilage little change.2 Comparison of the trachea after freezing storage organization and fresh tissue immunohistochemistry results, fresh trachea organizations: coronal slice in the trachea can be observed on the cortex to the base film organization has a small amount of dark brown stained cell body dendritic cell distribution in epithelial cell layer between the different shades of brown staining in the submucosal mixed glands are also deeply stained dendritic cells, airway long axis parallel to the base film can be observed slice a large number of dendritic cells, are woven into mesh, cartilage layer No positive staining. Trachea after cryopreservation: edema, swelling of dendritic cells, dendritic processes with pale staining, coloring remote processes is unclear, sparse cell distribution. OX-62 is a comparison of rat dendritic cell-specific markers, in the normal fresh group, the positive expression of a total of 8 cases, of which 6 cases of strong positive, positive in 1 case, weakly positive 1 case; in the freezer after 24 hours, its A total of eight cases of positive expression, including strong positive in 4 cases, positive in 2 cases, weakly positive in 2 cases, negative 0 cases; in the freezer after 10 days, the positive expression of a total of 8 cases, of which five cases of strong positive, positive in 2 cases, weakly positive in 1 case, negative 0 cases; in the freezer after 20 days, the positive expression of a total of 7 cases, of which strong positive in 1 case, positive in 3 cases, weakly positive in 4 cases, negative 0 cases; in the freezer after 30 days, the positive expression of A total of 5 cases, of which 0 cases of strong positive, positive in 1 case, weakly positive in 4 cases, negative in 3 cases; in the freezer after 60 days, the positive expression of a total of 1 case, in which strong positive 0 cases, 0 cases of positive, weakly positive one cases and negative in 7 cases; The results showed that, OX-62 in the freezer 24 hours, frozen 10 days, compared with the control group no significant difference (P>0.05), OX-62 in the freezer for 20 days, 30 days, 60 days group expressed significantly lower than the normal group (P<0.05), and the freezing time as the extension of its expression gradually decreasing.3 3H-TdR incorporation rates in the freezer before the measured value of 35.18±3.30CPM/mg, frozen after 24 hours 28.39±3.2CPM/mg, accounting for 80% of pre-frozen; frozen after 10 days of 26.28±2.62 CPM/mg , accounting for about 74% of pre-frozen; frozen after 20 days of 25.05±2.85 CPM/mg, accounting for about 71% of pre-frozen; frozen after 30 days of 24.85±3.01CPM/mg, accounting for 70.6% of pre-frozen; frozen after 60 days of 24.59±2.44CPM/mg, accounting for 65.2% before freezing.Conclusion:1 Frozen Gas pipe OX-62 expression decreased significantly statistically significant difference(≥20days), suggesting that cold gas storage pipe to reduce the immunogenicity of dendritic cells may be due to significantly reduced, its function and activity decreased, dendritic cells in the process of cold storage by the damage.2 3H-TdR in cell proliferation during the synthesis of the chromosome, cell death is not absorbed, it can be used as a sign of viability. By 3H-TdR incorporation test results analysis, cryopreservation trachea 24 hours after its incorporation in the significantly lower decrease gradually slowed since then.3 Comprehensive Our research results show that by detecting the different periods of frozen dendritic cells and dendritic cells, frozen after the change, and the vitality of tracheal cells and found that 60 days of tracheal frozen immunogenicity of the lowest, but the tracheal tissue energy is only 65% of pre-frozen, frozen 24-hour vitality of tracheal cells frozen before the 80%, but the immunogenicity is high; comprehensive comparison of the trachea 30 days after the freeze is not only a large degree of reduced immunogenicity, while to a large extent the preservation of the tracheal viability.