摘要
背景:细胞干燥保存的研究报道较多,组织器官是否适用于干燥保存,尚缺乏证据。目的:用海藻糖作为干燥保护剂,优化海藻糖载入大鼠皮肤的条件,探索皮肤干燥保存的可行性。设计、时间及地点:观察性实验,于2007-11/2008-11在西北大学生命科学学院组织工程实验室完成。材料:体质量150g左右成年SD大鼠。海藻糖由Sigma公司提供。方法:通过控制海藻糖浓度(50,300,500,800,1000mmol/L)、负载时间(0.5,4,7,9h)和温度[4℃、4～37℃(相转变)和37℃],优化海藻糖导入大鼠皮肤的条件。将负载海藻糖的皮肤置于含100g/L二甲基亚砜的DMEM冷冻液中孵育,程序冷冻仪以1℃/min的速率降温至-80℃后移入冷冻干燥机中进行冻干。将冻干皮肤水化复苏后分别用双醋酸羧基荧光素(CFDA)、四甲基偶氮唑盐(MTT)检测皮肤活性,并以新鲜和甲醛固定皮肤的吸光度值作为阳性和阴性对照。以苏木精-伊红染色和观察皮肤组织结构,并进行自体移植。主要观察指标:海藻糖在不同加载浓度、孵育温度和时间载入大鼠皮肤的情况。冻干皮肤水化后的形态学、组织学变化及其自体移植后存活情况。结果:海藻糖浓度小于800mmol/L,皮肤的海藻糖载入量随海藻糖浓度的升高而明显增(P<0.05),浓度大于800mmol/L,海藻糖载入量差异不显著。孵育温度为37℃组和相转变组皮肤海藻糖载入量明显高于4℃组(P<0.05),相转变组与37℃组差异无统计学意义。皮肤孵育4,7,9h的海藻糖载入量明显高于孵育0.5h的载入量(P<0.05),孵育时间为4,7,9h之间的海藻糖载入量差异不显著,但孵育7h后皮肤的海藻糖载入量不再增加。实验以海藻糖浓度为800mmol/L,孵育温度为37℃,孵育时间为7h为优化的负载条件处理大鼠皮肤。冻干皮肤玻璃化状态良好,呈半透明状。水化后能够恢复到新鲜皮肤的大小和色泽,组织结构和细胞形态与新鲜皮肤组织无差别。冷冻干燥皮肤的活性明显高于甲醛固定的大鼠皮肤(P<0.05)。冻干保存的皮肤水化后移植回自体大鼠,可存活长达13d。结论:海藻糖可作为干燥保护剂进行大鼠皮肤冷冻干燥保存,海藻糖浓度为800mmol/L,孵育温度为37℃,孵育时间为7h是海藻糖载入大鼠皮肤的最佳条件。
BACKGROUND： Researches on dry preservation of cells have been conducted. However, the dry preservation of tissue and organ remains poorly understood.
OBJECTIVE： To optimize the condition of introducing trehalose into rat skins, and investigate the feasibility of dry preservation of rat skins.
DESIGN, TIME AND SETTING： Observation-based experiment was performed at the Tissue Engineering Laboratory, College of Life Sciences, Northwest University from November 2007 to November 2008.
MATERIALS： SD adult rats, weighing 150 g, were selected. Trehalose was purchased from Sigma, USA.
METHODS： The condition of trehalose introduction was optimized by adjusting the trehalose concentration （50, 300, 500, 800, 1 000 mmol/L）, incubation period （0.5, 4, 7 and 9 hours） and incubation temperature [4 ℃, 4 %-37 ℃ （phase transition） and 37 %]. Trehatose loaded skins were placed in DMEM with 100 g/L dimethyl sulfoxide for 30 minutes, and cooled using a rate-controlled programmed freezer with the rate of 1 ℃ to 80 ℃ per minute, finally freeze-dried with freeze-dryer. The viability of skin grafts was evaluated by carboxyfluorescein diacetate, histological observation, and methyl thiazolyl tetrazolium viability assay. Absorbance of fresh and formalin fixed skins was separately regarded as positive and negative controls. HS staining was performed to observe skin structure, followed by autotransplantation.
MAIN OUTCOME MEASURES： Introduction of trehalose into rat skins in different trehalose concentrations, incubation temperatures and time; Morphological and histological observation after the skin being hydrated as well as the viability of hydrated and autotransplanted skins. RESULTS： The loading amount of trehalose in rat skins remarkably raised with the increasing trehalose concentrations till 800 mmol/L （P 〈 0.05）, and no apparent change was observed beyond 800 mmol/L （P 〉 0.05）. The uptake of trehalose in rat skin for 37 ℃ and phase transition groups was much higher than 4 ℃ groups （P 〈 0.05） but there was no significant difference between phase transition and 37 ℃ groups （P 〉 0.05）. The amount of trehalose in rat skin at 4, 7, 9 hours of incubation was notably higher than 0.5 hour group （P 〈 0.05）. However, no difference was found among 4, 7, and 9 hours groups, and trehalose loading efficiency in skins was no further increased after 7 hours of incubation （P 〉 0.05）. The viability of freeze-drying skins was markedly higher than the negative control （P 〈 0.05） group under the optimum conditions （800 mmol/L trehalose at 37 ℃ for 7 hours of incubation）, and morphology and histological observations were similar to fresh skin. The grafted skin could survive for 13 days after autotransplantation.
CONCLUSION： Trehalose concentration of 800 mmol/L, 37 ℃ as incubation temperature, and 7 hours of incubation are the best conditions for loading freeze-drying preservation of rat skin.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第41期8080-8084,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助项目(30870542)~~
