Investigation Of Cell Junction Associated Protein's Important Role And The Cryoprotectant During The Cryodamage Of Skin

Background and Objective: Cryopreservation has been extensively used in cryomedicine. Vitrification is considered as the best method for the cryopreservation of different organisms. However, how to improve the viability of the preserved skin is still unsatisfactory. It has reported that the reaction of tissues to hypothermy involves alterations in cell-cell and cell-matrix interactions. But the mechanism is not entirely cleared. As we know, the normal function of skin is highly dependent on the cell junction associated protein. The present study is designed to detect the realation between cryopreservation and cell junction associated protein, and make it more clear how they act on cryopreservation. The aim of the research is to provid scientific evidences for detecting effective elements by comparing several CPAS.Methods: In the first part of this thesis, the skins were stored in four different temperature: 4℃,-20℃,-80℃,-196℃ comparing with the fresh skin. After we observed the changes of cutaneous cells by microscopy, four associated proteins(vinculin,a-actinin) have been studied after 7 days, 14days and 21days.Then we analyzed the expression of these proteins by HE staining and immunohistochemical staining after different treatment, we used RT-PCR and Western blot to investigate the changes of the content of vinculin and α-actinin treated by different temperature.In the second part of this thesis, the cryopreserved skins were treated with DMSO/Propyleneglycol, DMSO, DMEM and trehalose/DMSO, comparing with the fresh skin. The viability of the skins was evaluated by using succinicdehydrogenase assay. Then the histological structure of different groups were observed and analyzed by pathological technology(include HE staining). Furthermor, we investigated the influence of trehalose on cell junction associated protein from the point of gene and protein through using RT-PCR and Western blot.Results: Cell junction associated protein was affected by temperature's alteration. In response to temperature downshift, the content of vinculin and α-actinin were all decreased. The mechanism underlyed these activities might associate with cutaneous cryodamage. 2. Variant alteration was appeared while stored the samples at different temperature. The skin which preserved at -196℃ was better than other groups. 3. Cell junction associated protein's expression of skin which preserved at -196℃ was downregulated by the time go on. 4.The results of electron microscope, immunohistochemical staining and the analysis of images shown cutaneous vinculin and α-actinin can be well-maintained through trehalose. 5. The results of viability revealed that skin cryopreserved with trehalose/DMSO showed indistinguishably from fresh skin, and better than other groups.Conclusion: In a word, conclusion can be made that the cryoinjury of skin might correlate with Cell junction associated protein , which play important roles in the mechanism of hypothermia preservation. Furthermore, trehalose is an available CPA which can improve the viability of the preserved skin. It ls suggested that cryopreservation protocols rutinely used in skin banks can be improved by combining trehalose with a permeating cryoprotectant.