| Background and Objective: With the development of cryobiology, practicehas found that, cryopreserved unicells have better viability than cryopreservedskin tissues. Researchers have gradually transfered the focus from cryoinjury onthe single cell to the integrity of the tissue. Worse result of cryopreserved tissuesis related to its complicated structure. Many kinds of cells are connected to makeup to a complicated ensemble by cell junction. And Cell junction maybe is oneof the key points which were injured by hypothermia. Desmosomes have thelargest number in epidermic cell junctions, and they can resist strong tension.Desmosome links cell tonofilaments together to make up to a net, which plays animportant roll to keep the integrity and viability of the tissue. The aim of thisstudy is to investigate the changes of desmosome treated by differenttemperatures and to look for a better croprotectant (CPA) for skin.Methods: In the first part of this experiment, the skins were stored in fourdifferent temperatures, 4℃,-20℃,-80℃,-196℃, comparing with the freshskin. After we observed the changes of cutaneous cells by microscopy,desmoglein1 and desmoglein2 have been studied after 7 days, 14days and21days.Then we analyzed the expression of these proteins by H.E.staining andimmunohistochemical staining after different treatments. We also observed theultrastructure of desmosome by electron microscope. Finally, we used RT-PCR toinvestigate the changes of gene level of desmoglein1 treated by four kinds oftemperatures.In the second part of this experiment, we applied two different cryoprotectants, DMSO/Propyleneglycol and trehalose/DMSO during cryopreserving skins. After 7 days and 21 days, the skin were rewarmed and compared with the fresh skin. The histological structure of different groups were observed and analyze by pathological technology (including H.E. staining, immunhistochemical staing and electron microscope). Then the viability of the skins was evaluated by using succinic dehydrogenase assay and oxygen consumption. Furthermore, we investigated the influence of the trehalose on desmoglein1 from the point of gene level through using RT-PCR.Results: The results of part one: 1.The expression regularity of desmoglein1 and desmoglein2 at different temperatures: comparing with the fresh skin,the content of two kinds of proteins all decreased in response to temperature downshift. But the skin which preserved at -196℃was better than other groups. 2. The effect of preservation time on the desmoglein1 at different temperatures: with time passing, the content of desmoglein1 at -196℃was decreased slower than other groups. 3. The change of desmosome at different temperatures: the ultrastructure of desmosome was distorted more or less at different temperatures, but at -196℃desmosome was almost same with the fresh group.The results of part two: 1. With time going on,the result of electron microscope,immunohistochemical staining and RT-PCR showed the ultrastructure of desmosome and desmoglein1 can be well-protected by trehalose/DMSO. 2. According to SDH and oxygen consumption assay, the viability of skins preserved by trehalose/DMSO is better than preserved by DMSO/Propyleneglycol.Conclusion: In a word, the conclusion can be made that cryoinjury of skin might correlate with desmosome and associated proteins, which shows that cell junction may play an important role via desmosome in the mechanism of cryopreservation. Furthermore, trehalose improves the viability of skins, which may be related with protecting desmosome proteins. |
PDF Full Text Request