本文選題：抗氧化劑 + 人顆粒脂肪組織　； 參考：《南華大學(xué)》2015年碩士論文

【摘要】：背景:自體脂肪是一種理想的填充材料,幾乎可用于全身各個部位的軟組織缺損治療,在整形外科中廣泛應(yīng)用。有研究報道,采用取自上臂小塊脂肪組織游離移植填充下眶緣軟組織缺損獲得了良好的美容效果,認為抽取自體脂肪進行注射移植這種方法是值得推廣的。然而目前填充移植后保存的脂肪組織體積最終只有注射時的20-30%左右。為了達到滿意的填充效果,臨床上常常需要進行再次或者多次填充移植。反復(fù)的脂肪抽取操作既增加了醫(yī)生的工作量,又增加了患者為取材而吸脂的手術(shù)痛苦和手術(shù)風(fēng)險,并造成較高的費用。在此背景下如能通過一次吸脂手術(shù)獲得足夠的脂肪組織,同時將獲取的脂肪進行體外長期冷凍儲存,在需要時復(fù)溫即可使用,是廣大醫(yī)生和患者的迫切愿望,同時也具有重要的臨床應(yīng)用價值和經(jīng)濟價值,冷凍過程最關(guān)鍵的是冷凍保護劑的選用。但目前國內(nèi)外關(guān)于人顆粒脂肪組織冷凍保護劑的研究尚少,用于凍存其他細胞或組織時的冷凍保護劑是否可保持冷凍保存脂肪組織中細胞的完整結(jié)構(gòu)和活力的研究還鮮見報道。通常認為在冷凍保存過程中細胞損傷主要來自兩方面:一是降溫時細胞內(nèi)生成粗大的樹枝狀冰晶直接破壞細胞結(jié)構(gòu);另一方面則是細胞在降溫過程中氧化應(yīng)激產(chǎn)生過多過氧化物,使細胞活力受損,加速了細胞的凋亡。目的:本次研究在基礎(chǔ)凍存液中添加目前臨床較常使用的抗氧化劑來進行人顆粒脂肪組織的凍存,觀察復(fù)溫后的細胞結(jié)構(gòu)代謝、及細胞活性等方面的變化,初步探討添加抗氧化劑是否更有利于人顆粒脂肪組織的凍存,為自體脂肪注射移植實現(xiàn)“一次凍存,長期使用”的目標和提高凍存后人顆粒脂肪組織移植的成活率,減少患者的痛苦和醫(yī)生的工作量,提供相關(guān)理論支持和基礎(chǔ)實驗依據(jù)。方法:以配制滲透性玻璃化冷凍保護劑(生理鹽水+8%二甲基亞砜)為基礎(chǔ)液,分別以不同濃度的抗氧化劑超氧化物歧化酶(100,200,400,600 IU/m L),谷胱甘肽(1,5,10,15 mmol/L)和生育酚(維生素E,1,1.5,2,2.5 mg/m L)配制凍存液;再與抽取獲得經(jīng)分離提純后的人顆粒脂肪組織混合均勻,置于-4℃冰箱中凍存2個月后取出;復(fù)溫后通過組織石蠟包埋切片HE染色觀察細胞結(jié)構(gòu)是否完整,組織冰凍切片油紅O染色觀察脂肪細胞中脂滴形態(tài),以及ROS檢測盒熒光染色流式細胞術(shù)分析細胞內(nèi)ROS含量,碘化丙錠(PI)染色流式細胞術(shù)分析細胞凋亡,觀察分析添加抗氧化劑凍存后人顆粒脂肪組織中細胞的基本結(jié)構(gòu)、代謝及活力改變。結(jié)果:HE染色顯示,經(jīng)單純PS+8%DMSO基礎(chǔ)凍存液凍存的人顆粒脂肪組織復(fù)溫后組織結(jié)構(gòu)不均勻,鏡下可見大片囊樣脂池和壞死區(qū)。而100-600 IU/m L SOD(或1-15 mmol/L GSH或1-2.5 mg/m L VE)+PS+8%DMSO凍存組復(fù)溫后脂肪組織結(jié)構(gòu)較均勻,大的囊樣脂池形成較少。油紅O染色顯示,經(jīng)單純PS+8%DMSO基礎(chǔ)凍存液凍存的脂肪組織復(fù)溫后脂肪組織中脂滴大部分破裂,少見藍色胞核,而100-600 IU/m L SOD(或1-15 mmol/L GSH或1-2.5 mg/m L VE)+PS+8%DMSO凍存的可見部分藍染胞核,紅色脂滴,但形態(tài)欠佳。ROS檢測盒熒光標記流式細胞儀分析數(shù)據(jù)成圖顯示,單純PS+8%DMSO基礎(chǔ)凍存液組顆粒脂肪組織的熒光含量峰值最高,多于其他實驗組(P0.05)。以不同濃度SOD(或GSH或VE)+PS+8%DMSO為冷凍保護劑凍存顆粒脂肪組織,8周后復(fù)溫,通過PI單染的流式細胞術(shù)測定細胞凋亡,結(jié)果顯示,SOD+PS+8%DMSO凍存組凋亡細胞曲線峰均低于單純PS+8%DMSO基礎(chǔ)凍存液凍存組(P0.05)。結(jié)論:1.用常規(guī)凍存液含8%二甲基亞砜生理鹽水在-4℃凍存人顆粒脂肪組織2個月后解凍復(fù)溫,脂肪組織在結(jié)構(gòu)完整性、細胞內(nèi)脂滴含量明顯下降,細胞凋亡水平增加。2.在常規(guī)凍存液添加不同濃度的抗氧化劑超氧化物歧化酶、還原性谷胱甘肽或維生素E后,在-4℃凍存人顆粒脂肪組織2個月后解凍復(fù)溫,脂肪組織的結(jié)構(gòu)完整性和細胞內(nèi)脂滴含量有不同程度的改善,細胞內(nèi)活性氧水平和細胞凋亡水平明顯降低。3.在本次實驗濃度范圍下,超氧化物歧化酶、還原性谷胱甘肽或維生素E保護細胞的凍融損傷呈劑量效應(yīng)依賴性。
[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.

【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R622

【參考文獻】

相關(guān)期刊論文 前10條

1 李志超;馮云;;改善卵母細胞冷凍損傷的研究進展[J];中國優(yōu)生與遺傳雜志;2014年08期

2 仇侃敏;吳蒙;梁耀蟬;羅婕姝;謝紅炬;;不同凍存液保存的顆粒性脂肪組織活力及移植成活率分析[J];基礎(chǔ)醫(yī)學(xué)與臨床;2014年04期

3 韓鵬;王尚乾;唐敏;徐楊;張煒;;維生素E對人精子凍融氧化應(yīng)激損傷的保護作用[J];中華男科學(xué)雜志;2014年02期

4 王晶;馬鵬程;柳立軍;;天然抗氧化劑在人精液冷凍保存中的應(yīng)用[J];國際生殖健康/計劃生育雜志;2013年06期

5 賈永宏;馬國際;王春偉;洪潔峗;胡建宏;;抗氧化劑對家畜精液冷凍保存的應(yīng)用研究進展[J];家畜生態(tài)學(xué)報;2013年01期

6 謝紅炬;鄧穎;李明;羅惠中;陳碾;;自體純化冷凍微粒脂肪注射移植修復(fù)面部皮膚軟組織老化性萎縮凹陷[J];中國組織工程研究;2012年05期

7 劉乃軍;管延萍;王艷;黃金井;;濕性脂肪干細胞輔助自體脂肪移植術(shù)[J];中國美容醫(yī)學(xué);2011年07期

8 鄧穎;謝紅炬;;自體脂肪細胞注射移植研究進展[J];現(xiàn)代醫(yī)藥衛(wèi)生;2011年09期

9 劉元剛;張晨;程欲;劉樹滔;饒平凡;;局部應(yīng)用PTD-SOD、SOD對小鼠皮膚創(chuàng)傷的抗氧化應(yīng)激損傷保護效果及其比較[J];中國實驗動物學(xué)報;2010年06期

10 付冰川;高建華;魯峰;李R，

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