抗氧化剂对冷冻人颗粒脂肪组织的保护作用

发布时间：2018-04-26 23:23

本文选题：抗氧化剂 + 人颗粒脂肪组织　；参考：《南华大学》2015年硕士论文

【摘要】：背景:自体脂肪是一种理想的填充材料,几乎可用于全身各个部位的软组织缺损治疗,在整形外科中广泛应用。有研究报道,采用取自上臂小块脂肪组织游离移植填充下眶缘软组织缺损获得了良好的美容效果,认为抽取自体脂肪进行注射移植这种方法是值得推广的。然而目前填充移植后保存的脂肪组织体积最终只有注射时的20-30%左右。为了达到满意的填充效果,临床上常常需要进行再次或者多次填充移植。反复的脂肪抽取操作既增加了医生的工作量,又增加了患者为取材而吸脂的手术痛苦和手术风险,并造成较高的费用。在此背景下如能通过一次吸脂手术获得足够的脂肪组织,同时将获取的脂肪进行体外长期冷冻储存,在需要时复温即可使用,是广大医生和患者的迫切愿望,同时也具有重要的临床应用价值和经济价值,冷冻过程最关键的是冷冻保护剂的选用。但目前国内外关于人颗粒脂肪组织冷冻保护剂的研究尚少,用于冻存其他细胞或组织时的冷冻保护剂是否可保持冷冻保存脂肪组织中细胞的完整结构和活力的研究还鲜见报道。通常认为在冷冻保存过程中细胞损伤主要来自两方面:一是降温时细胞内生成粗大的树枝状冰晶直接破坏细胞结构;另一方面则是细胞在降温过程中氧化应激产生过多过氧化物,使细胞活力受损,加速了细胞的凋亡。目的:本次研究在基础冻存液中添加目前临床较常使用的抗氧化剂来进行人颗粒脂肪组织的冻存,观察复温后的细胞结构代谢、及细胞活性等方面的变化,初步探讨添加抗氧化剂是否更有利于人颗粒脂肪组织的冻存,为自体脂肪注射移植实现“一次冻存,长期使用”的目标和提高冻存后人颗粒脂肪组织移植的成活率,减少患者的痛苦和医生的工作量,提供相关理论支持和基础实验依据。方法:以配制渗透性玻璃化冷冻保护剂(生理盐水+8%二甲基亚砜)为基础液,分别以不同浓度的抗氧化剂超氧化物歧化酶(100,200,400,600 IU/m L),谷胱甘肽(1,5,10,15 mmol/L)和生育酚(维生素E,1,1.5,2,2.5 mg/m L)配制冻存液;再与抽取获得经分离提纯后的人颗粒脂肪组织混合均匀,置于-4℃冰箱中冻存2个月后取出;复温后通过组织石蜡包埋切片HE染色观察细胞结构是否完整,组织冰冻切片油红O染色观察脂肪细胞中脂滴形态,以及ROS检测盒荧光染色流式细胞术分析细胞内ROS含量,碘化丙锭(PI)染色流式细胞术分析细胞凋亡,观察分析添加抗氧化剂冻存后人颗粒脂肪组织中细胞的基本结构、代谢及活力改变。结果:HE染色显示,经单纯PS+8%DMSO基础冻存液冻存的人颗粒脂肪组织复温后组织结构不均匀,镜下可见大片囊样脂池和坏死区。而100-600 IU/m L SOD(或1-15 mmol/L GSH或1-2.5 mg/m L VE)+PS+8%DMSO冻存组复温后脂肪组织结构较均匀,大的囊样脂池形成较少。油红O染色显示,经单纯PS+8%DMSO基础冻存液冻存的脂肪组织复温后脂肪组织中脂滴大部分破裂,少见蓝色胞核,而100-600 IU/m L SOD(或1-15 mmol/L GSH或1-2.5 mg/m L VE)+PS+8%DMSO冻存的可见部分蓝染胞核,红色脂滴,但形态欠佳。ROS检测盒荧光标记流式细胞仪分析数据成图显示,单纯PS+8%DMSO基础冻存液组颗粒脂肪组织的荧光含量峰值最高,多于其他实验组(P0.05)。以不同浓度SOD(或GSH或VE)+PS+8%DMSO为冷冻保护剂冻存颗粒脂肪组织,8周后复温,通过PI单染的流式细胞术测定细胞凋亡,结果显示,SOD+PS+8%DMSO冻存组凋亡细胞曲线峰均低于单纯PS+8%DMSO基础冻存液冻存组(P0.05)。结论:1.用常规冻存液含8%二甲基亚砜生理盐水在-4℃冻存人颗粒脂肪组织2个月后解冻复温,脂肪组织在结构完整性、细胞内脂滴含量明显下降,细胞凋亡水平增加。2.在常规冻存液添加不同浓度的抗氧化剂超氧化物歧化酶、还原性谷胱甘肽或维生素E后,在-4℃冻存人颗粒脂肪组织2个月后解冻复温,脂肪组织的结构完整性和细胞内脂滴含量有不同程度的改善,细胞内活性氧水平和细胞凋亡水平明显降低。3.在本次实验浓度范围下,超氧化物歧化酶、还原性谷胱甘肽或维生素E保护细胞的冻融损伤呈剂量效应依赖性。
[Abstract]:Background: autologous fat is an ideal filling material which can be used in the treatment of soft tissue defects in all parts of the body. It is widely used in plastic surgery. The method of transplantation is worth promoting. However, the volume of fat tissue preserved after filling is only about 20-30% at the time of injection. In order to achieve satisfactory filling effect, it is often necessary to carry out re or multiple filling transplants. Repeated fat extraction operations increase the workload of the doctor and increase the patient's workload. In this context, it is the urgent desire of the vast majority of the medical students and patients. The key to the clinical application and economic value is the selection of cryopreservation agent. But at present, there are few studies on the cryopreservation agent of human granular fat tissue at home and abroad. Whether the cryopreservation agent for cryopreservation of other cells or tissues can maintain the complete structure and vitality of the cryopreservation of the cells in the freeze-preserved adipose tissue It is also rarely reported that cell damage mainly comes from two aspects in the process of cryopreservation: one is that the cell structure is destroyed by large dendritic ice crystals during cooling, and on the other hand, oxidative stress produces peroxides in the process of cooling, causing cell vitality to damage and accelerating cell apoptosis. In this study, we added the current commonly used antioxidants to the cryopreservation of human granular adipose tissue, observed the changes in cell structure metabolism and cell activity after rewarming, and preliminarily discussed whether the addition of antioxidants is more beneficial to the cryopreservation of human fat tissue and the autotransplantation of fat. The objective of "one frozen storage, long term use" and to improve the survival rate of the transplantation of fat tissue after cryopreservation, reduce the sufferings of the patients and the workload of the doctors, provide relevant theoretical support and basic experimental basis. Methods: Based on the preparation of osmotic vitrification cryopreservation agent (+8% two methyl sulfoxide) as the base liquid, the difference is different. The concentration of antioxidants superoxide dismutase (100200400600 IU/m L), glutathione (1,5,10,15 mmol/L) and tocopherol (vitamin E, 1,1.5,2,2.5 mg/m L) prepared from the cryopreservation liquid, and then mixed evenly with the extracted human granular adipose tissue after separation and purification, and removed in the freezer for 2 months at -4 C; after rewarming, the paraffin tissue was formed by tissue paraffin. HE staining was used to observe the integrity of cell structure, tissue frozen section oil red O staining to observe the lipid droplet morphology in adipocytes, and ROS detection box fluorescence staining flow cytometry to analyze the intracellular ROS content. PI staining flow cytometry was used to analyze cell apoptosis. The basic structure, metabolism and vitality change of the cells in the fabric. Results: HE staining showed that the tissue structure was not uniform after rewarming of the human granular adipose tissue frozen through the pure PS+8%DMSO basic cryopreservation solution, and large sac like fat pools and necrotic areas were visible under the microscope. 100-600 IU/m L SOD (or 1-15 mmol/L GSH or 1-2.5 mg/m L VE) +PS+8%DMSO cryopreservation group after rewarming The fat tissue of the fat tissue was relatively small. The oil red O staining showed that the fat tissue in the adipose tissue was mostly ruptured after the rewarming of the frozen storage liquid of the simple PS+8%DMSO base, and the blue nucleus was rare, while the 100-600 IU/m L SOD (or 1-15 mmol/L GSH or 1-2.5 mg/m L VE) was seen in the visible part of the blue dyed nucleus of the +PS+8%DMSO. Red lipid droplets, but the.ROS detection box fluorescence labeling flow cytometer analysis data showed that the peak fluorescence content of the granular fat tissue in the simple PS+8%DMSO base frozen liquid group was the highest, more than that of the other experimental groups (P0.05). SOD (or GSH or VE) +PS+8% DMSO was used as the cryopreservation agent to freeze the granular adipose tissue, and the temperature was recovered after 8 weeks. The cell apoptosis was measured by PI single dye flow cytometry. The results showed that the peak of apoptotic cells in the SOD+PS+8%DMSO cryopreservation group were lower than that of the simple PS+8%DMSO base cryopreservation group (P0.05). Conclusion: 1. the normal saline containing 8% two methyl sulfoxide in normal saline was thawing and rewarming after 2 months of -4 cryopreservation of human granular adipose tissue, and the adipose tissue was in the knot. The content of lipid droplets in cells decreased significantly, and the level of cell apoptosis increased by.2. in the conventional cryopreservation liquid, after adding different concentrations of antioxidant superoxide dismutase, reduced glutathione or vitamin E, rewarming at 2 months after the cryopreservation of human granular adipose tissue at -4 C, the structural integrity of adipose tissue and the content of intracellular lipid droplets. With different degrees of improvement, the level of intracellular reactive oxygen species (ROS) and the level of cell apoptosis significantly decreased.3. under this experimental concentration. The freezing thawing damage of superoxide dismutase (SOD), reduced glutathione or vitamin E protected cells was dose-dependent.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R622

【参考文献】