The Effect Of Cryopreservation On β₁integrin And Associated Proteins Of Skin And The Intervention Study

Background and Objective: With the development of cryobiology in recent years, researchers have gradually transferred the focus from cryoinjury on the single cell to the integrity of the cryopreserved tissue. As the microenvironment of cells, any changes of extracellular matrix may influence the biological character and function of the whole tissue via cell membrane. The disruption of stroma cells whose viability is high in the epidermis would destroy the total viability of the free skin graft. It has been found that there exist an important glycoprotein β₁integrin on the membrane of the stroma cells, which is also the receptor of many extracellular matrix proteins. According to contacting with talin, β₁integrin acts as the bridge between cell and extracellular matrix, which may play an important role in keeping the integrity and normal function of the cell membranes.Methods: In the first part of this experiment, the skins were stored in four different temperatures,4℃,-20℃,-80℃,-196℃,comparing with the fresh skin.After we observed the changes of cutaneous cells by microscopy, β₁integrin and associated proteins(laminin and talin)have been studied after 7 days,14days and 21days.Then we analyzed the expression of these proteins by H.E.staining and immunohistochemical staining after different treatments. We also observed the ultrastructure of basal lamina by electron microscope. Finally ,we used RT-PCR to investigate the changes of gene level of β₁integrin treated by four kinds of temperatures.In the second part of this experiment, we applied two different cryoprotectants,DMSO/Propyleneglycol and trehalose/DMSO,during cryopreserving skins.After 7 days and 14 days,the skins 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 theskins was evaluated by using SDH assay. Furthermore, we investigated the influence of the trehalose on β₁integrin from the point of gene level through using RT-PCR.Results: The results of part one:1.The expression regularity of β₁integrin ,laminin and talin at different temperatures: comparing with the fresh skin,the content of three 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 β₁integrin at different temperatures: with time passing, the content of β₁integrin at -196℃ was decreased slower than other groups. 3. The change of basal lamina at different temperatures: the ultrastructure of basal lamina was distoried more or less at different temperatures, but at -196℃ basal lamina 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 basal lamina and β₁integrin can be well-protected by trehalose/DMSO. 2. According to SDH assay, the viability of skins preserved by trehalose/DMSO is better than preserved by DMSO/Propyleneglycol.Conclusion: In a word ,conclusion can be made that cryoinjury of skin might correlated with β₁integrin and associated proteins(laminin and talin),which show that extracellular matrix may play an important role via β₁integrin and talin in the mechanism of cryoinjury. Furthermore, trehalose improve the viability of skins,which may related with protecting β₁integrin .