去分化脂肪细胞的细胞生物学研究

发布时间：2018-03-26 07:30

本文选题：脂肪细胞　切入点：脂肪组织来源干细胞　出处：《南方医科大学》2008年硕士论文

【摘要】： 背景组织工程技术的出现为临床上彻底解决组织缺损等难题提供了一条革命性的途径。然而如何获得来源广泛、数量充足、功能良好的细胞,是解决种子细胞问题的关键。近年来,吸脂来源的人脂肪组织来源的干细胞(Adipose derived stemcells,ASCs)成为当今干细胞研究领域的一大热点。但ASCs是位于人脂肪组织内的一个混杂细胞群体。只有大约40%的细胞能向脂肪细胞分化。因此,ASCs并不能满足脂肪组织工程的发展需要。寻找一种分离容易、增殖能力强、成脂分化率高的更优秀的种子细胞仍然是研究热点。脂肪组织内含有多种不同的细胞,主要包含有成熟脂肪细胞、血管内皮细胞、成纤维细胞、组织细胞和干细胞等。大量的脂质聚集使脂肪细胞具有的漂浮性,导致细胞难于体外培养,因此对该细胞的特性研究很少。脂肪细胞由此被认为是处于分化终末期,缺乏增殖能力的一种细胞。国外有研究表明,成熟脂肪细胞在体外培养的过程中,能发生脂质分解变化,重新启动增殖机制,开始分裂增生。我们把这种生理转变称之为脂肪细胞去分化。脂肪细胞去分化将开创细胞生理学一个令人惊喜的领域,改变人们对于组织再生能力的许多观点。然而,关于去分化脂肪细胞的细胞生物学特性研究还很少。本实验中我们尝试从脂肪抽吸物的脂质部分分离纯的成熟脂肪细胞,诱导成熟脂肪细胞去分化,获得去分化脂肪细胞(Dedifferentiated adipocytes,DA)。同时对DA和脂质部分来源的ASCs进行生长动力学、形态学、表面标志物、分化能力和成脂分化率五个方面的鉴定比较。为获得、研究DA提供新的途径,并首次为DA作为脂肪组织工程种子细胞的应用提供实验依据。目的: 1、探索从人脂肪抽吸物中脂质部分分离、提纯成熟脂肪细胞,体外培养,以及诱导其去分化,获得去分化脂肪细胞的方法。 2、通过与ASCs的比较,于形态学、生长动力学、表面标记物、分化能力及成脂分化率五个方面对DA进行研究,为其广泛应用于组织工程提供实验依据。方法: 自成人吸脂术后抽吸物提取成熟脂肪细胞及ASCs,天花板贴壁培养法诱导成熟脂肪细胞去分化,观察细胞形态变化,获得DA。相同的条件下,MTT比色法比较DA、ASCs活性并绘制细胞生长曲线;流式细胞仪鉴定DA、ASCs表面分子的表达;油红O染色、茜素红染色、阿尔辛蓝染色分别鉴定DA、ASCs成脂分化、成骨分化、成软骨分化能力:大体镜下观察、油红O染色分光光度法、油红O染色细胞计数法比较DA、ASCs成脂分化能力。结果: 人成熟脂肪细胞在体外培养环境下能去分化为成纤维细胞状DA;MTT比色法测细胞活性:DA、ASCs都有很强的增殖能力,两者无显著性差异;流式细胞仪测定:DA、ASCs中HLA-ABC、CD29、CD44都为阳性,CD45、CD34、CD106都为阴性;成骨分化两周,茜素红染色可见DA、ASCs内出现红色钙盐沉积;成软骨分化两周,阿尔辛蓝染色可见DA、ASCs内软骨基质沉积;成脂分化两周,油红O染色可见DA、ASCs内出现红色脂滴;大体镜下观察显示DA脂滴聚集能力显著大于ASCs;油红O染色分光光度法显示DA在诱导第4天后出现显著性脂滴聚集,ASCs在诱导第10天后才出现显著性脂滴聚集,诱导第12天后DA的吸光值大于ASCs的吸光值,出现显著性差异;油红O染色细胞计数法示DA的平均成脂分化率约为65%,ASCs的约为35%,两者有显著性差异。结论: 从成人脂肪抽吸物脂质部分中可以分离、提纯得到大量的成熟脂肪细胞。成熟的脂肪细胞在体外培养条件下可成为DA,DA与ASCs在细胞的生长动力学、形态学、表面标志物和分化能力等方面具有相似的特征,具有很强的增殖活性,表达部分干细胞特征性表面蛋白,有成骨、成软骨分化能力及强大的成脂分化能力,有望成为脂肪组织工程优秀的种子细胞。
[Abstract]:background
The emergence of tissue engineering technology provides a revolutionary way for solving the problems of tissue defects in clinical practice. However, how to get a wide range of cells with sufficient quantity and good function is the key to solve the problem of seed cells.
In recent years, liposuction from human adipose tissue derived stem cells (Adipose derived stemcells, ASCs) has become a hot research field of stem cells. But ASCs is a mixed cell population in human adipose tissues. Only about 40% of the cells to adipose cell differentiation. Therefore, ASCs does not meet the the need of the development of adipose tissue engineering. Looking for an easy separation, strong proliferation ability, a more excellent seed cell fat high differentiation rate is still the focus of research.
Contains a variety of different cells in adipose tissue, are mainly composed of mature fat cells, vascular endothelial cells, fibroblasts, cells and tissue stem cells. A large number of lipid accumulation in fat cells with the floating cells in vitro, resulting in difficult, so little research on the characteristics of the cell. The fat cells is thus considered is in the terminal stage of differentiation, the lack of a cell proliferation. Foreign studies have shown that mature adipocytes in vitro, lipolysis changes can occur, restart the proliferation mechanism, begin to proliferate. The physiological changes known to differentiate into fat cells.
Fat cells to differentiation of cell physiology will create a surprise for many people, change view regeneration ability of organization. However, the research on the biological characteristics of cells to adipocyte differentiation are rare. In this experiment we attempt to separate pure mature fat cells from the lipid portion of liposuction, induction of mature fat cells to get to the differentiation of adipocyte differentiation (Dedifferentiated, adipocytes, DA). At the same time, DA and ASCs are the source of the lipid portion of the growth kinetics, morphology, surface markers, differentiation and adipogenic differentiation rate of identification in five aspects. In order to obtain and provide a new way of DA, and for the first time for the application of DA as adipose tissue engineering seed cells and provide experimental basis.
Objective:
1, we need to explore the way to isolate and purify mature adipocytes from human liposuction, to induce dedifferentiation, and to get dedifferentiated adipocytes.
2, by comparing with ASCs, we studied DA in five aspects: morphology, growth kinetics, surface markers, differentiation ability and adipogenic differentiation rate, providing experimental evidence for its wide application in tissue engineering.
Method:
Since adults after liposuction aspirates from mature fat cells and ASCs ceiling adherent induced dedifferentiation of mature adipocytes were cultured. The morphological changes were observed for DA. under the same conditions, MTT colorimetric method DA, ASCs activity and cell growth curve; flow cytometry identification DA surface expression of ASCs molecules; oil red O staining, alizarin red staining, alcian blue staining were identified as DA ASCs, adipogenic differentiation, osteogenic differentiation and chondrogenic differentiation ability: observed under microscope oil red O staining and spectrophotometry, oil red O staining cell count is compared with DA, ASCs and adipogenic differentiation ability.
Result:
Human mature adipocytes in vitro environment can differentiate into fibroblast like DA; MTT cell activity assay: DA and ASCs had strong proliferation ability, no significant difference; flow cytometry: DA, ASCs HLA-ABC, CD29, CD44 were positive for CD45. CD34, CD106 were negative; osteogenic differentiation for two weeks, alizarin red staining showed red DA, calcium salt deposition within ASCs; chondrogenic differentiation for two weeks, alcian blue staining, DA, ASCs in cartilage matrix deposition; adipogenic differentiation for two weeks, oil red O staining showed DA, ASCs appeared in red lipid droplets; general microscopy showed that DA lipid droplet aggregation ability was significantly greater than ASCs; oil red O staining spectrophotometric method showed that DA in fourth days after the induction of significant lipid droplet aggregation, ASCs appeared significant lipid droplet aggregation in tenth days after induction, Twelfth days after the induction of DA light absorption value is greater than ASCs value, there was significant difference The oil red O staining cell count method showed that the average fat differentiation rate of DA was about 65%, and that of ASCs was about 35%, and there was a significant difference between them.
Conclusion:
Can be isolated from adult adipose aspirates lipid fraction, purified mature fat cells. Mature adipocytes into DA in vitro, DA and ASCs on the growth kinetics, cell morphology, surface markers and differentiation capacity were similar characteristics, with strong proliferation, expression some characteristics of stem cell surface protein, osteogenic, chondrogenic differentiation ability and strong adipogenic differentiation ability, is expected to become fat seed cells for tissue engineering of excellent.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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