【作者】 李东杰；

【导师】 贾晓明；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科学烧伤， 2008， 硕士

【摘要】 研究背景及目的:随着低温生物医学的发展,低温保存的实践发现,当组织细胞以单细胞悬液的形式冷冻保存时,可以保持比保存皮肤组织更高的活力。研究者目光已逐渐从低温损伤对单细胞的影响转移到对组织完整性的破坏上。组织保存的效果较差可能与组织复杂的结构有关,而组织复杂的结构主要是靠细胞间连接将多种细胞联系在一起形成成一个整体。细胞连接是组织低温损伤的关键部位之一。表皮角质形成细胞间的连接以桥粒(desmosome)的分布最多。桥粒具有很强的抗牵张力,通过相邻细胞间张力细丝网的机械性连接,形成一连续的结构网。对于维持细胞骨架和组织完整性及其活性方面发挥着重要意义。本课题旨在研究皮肤组织低温储存后桥粒的关键跨膜成分桥粒芯糖蛋白1(desmoglein1,Dsg1)、桥粒芯糖蛋白2(desmoglein2,Dsg2)的改变,同时继续探讨海藻糖作为低温保护剂的研究,通过其对皮肤低温损伤的干预,观察desmoglein1、desmoglein2改变,进一步揭示皮肤低温损伤机制并为完善海藻糖作为低温保护剂有效成分提供实验依据。方法:在第一部分实验中,取烧伤整形术后(4小时内)所余正常人皮肤组织皮片,采用不同的温度组(4℃组、-20℃组、-80℃组、-196℃组)进行保存,设新鲜皮肤组为对照组。分别在不同的时间点进行实验观察,首先通过H.E.染色和免疫组织化学染色两种病理方法在光镜下观察表皮细胞形态和桥粒跨膜蛋白的变化,采用ImagePro5.0图像分析软件进行图像分析,研究指标为desmoglein1、desmoglein2;其次,采用透射电镜观察桥粒形态结构的变化,并应用RT-PCR方法检测不同温度保存后desmoglein1基因水平表达规律。以证实桥粒为低温损伤的关键位点,推测其损伤的原理,寻觅更多关于抗低温损伤的机理。在第二部分实验中,我们继以往海藻糖(trehalose)对皮肤组织细胞骨架成分及整合素保护作用的基础之上,进一步采用免疫组织化学方法观察海藻糖对desmoglein1、desmoglein2表达的影响,RT-PCR方法观察了干预条件下desmoglein1的基因表达,应用透射电镜观察相应的细胞连接桥粒的形态结构变化,并分别用琥珀酸脱氢酶(SDH)定量检测法和氧耗量测定法比较两种不同低温保护剂组合对表皮活力的影响。结果:从第一部分实验结果发现:1.不同低温储存皮肤后桥粒芯糖蛋白表达规律:4℃、-20℃、-80℃、-196℃组desmoglein1、desmoglein2水平均有不同程度的下降。与新鲜对照组相比,4℃组desmoglein1、desmoglein2出现显著降低,而-196℃组下降程度不明显。2.不同储存时间对desmoglein1、desmoglein2表达的影响:其中4℃组储存情况下,各项指标随时间变化下降最为明显,其次为-20℃组,而-196℃组桥粒芯糖蛋白水平均略有下降,-80℃组蛋白含量则介于-196℃组、-20℃组之间。3.不同低温储存皮肤后表皮细胞间桥粒的变化规律:通过电镜观察发现-196℃组桥粒结构清晰可见,保持完好;4℃组可见桥粒数量明显减少,结构模糊不清或碎裂,-80℃组、-20℃组桥粒变化介于上述两者之间,且-80℃组比-20℃组桥粒保存更为完整。第二部分实验结果发现:1.通过免疫组织化学观察可见,在第7天时,T/D、D/P组desmoglein1表达量与新鲜组相似。随时间延长,T/D组表现出了较传统保护剂组对desmoglein1保护更为优势的作用。T/D、D/P组desmoglein2表达量与新鲜组相比无显著差别。从RT-PCR实验结果观察,经过低温储存1周后,T/D、D/P组desmoglein1表达量与新鲜组近似,随时间延长D/P组表达相对减弱。2.通过比较活力,发现随储存时间的延长,各组皮片活力呈不同程度的下降。统计分析结果显示,储存时间为1周时,T/D组、D/P组间活力存在差异,而储存时间为3周时,T/D组和D/P组间随时间延长T/D组皮肤活力优于D/P组。结论:细胞连接桥粒结构在皮肤低温损伤中发生了较为明显的变化,结合桥粒在结构上与细胞骨架的密切关系以及既往对细胞骨架在低温损伤中的研究基础分析,桥粒可能是皮肤低温损伤的重要位点,并参与并介导细胞骨架对皮肤组织的低温损伤中发挥着重要的作用。在传统低温保护剂冲添加海藻糖后,皮肤活力得以提高的原因体现在多方面,对桥粒跨膜蛋白的保护作用是活力得以提高的重要原因之一。更多还原

【Abstract】 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.更多还原