脂肪组织来源干细胞单克隆培养技术及其表面抗原的研究

发布时间：2018-09-05 13:23

【摘要】： 背景: 组织工程技术以少量的种子细胞经体外扩增后与生物材料结合,修复较大的组织或器官缺损,重建缺损部位的生理功能,为临床上解决组织缺损等难题提供了一条新途径,近来受到广泛关注。如何获得来源广泛、充足的种子细胞是此项技术的关键。成体干细胞来源广泛、功能良好,是种子细胞的研究重点。成体干细胞可来源于骨髓、脂肪、皮肤、肌肉等各类组织,其中骨髓基质干细胞(BMSCs),发现早、取材方便、创伤小,被研究的最多。随着研究的普及和深入,脂肪组织来源干细胞(ADSCs)逐渐被人们认识,与BMSCs相比,ADSCs在体内分布更广,可利用的细胞量更多。ADSCs自1994年被发现以来已证实能向多种类型的细胞分化,并且具有取材方便、创伤小而细胞获得量大、病人易于接受的优势,在组织工程的研究和应用中显示出了巨大的潜力。目前普遍认为,常用方法分离出的ADSCs是由多种分化潜能不同的细胞亚群以及大量的混杂细胞组成的混合群体,其中只有部分能够形成脂肪细胞,因而必然会影响其构建脂肪组织的成功率。如何提高从脂肪组织中提取干细胞的纯度和寻找确认干细胞的相关标记等问题一直是目前急于解决的难题之一。虽然对于ADSCs的表面抗原的研究已有诸多报道,但有关ADSCs的特异性表面标志仍无统一认识,对于特定分化潜能和标志物表达之间是否存在关联至今尚未见有报道。此外,有关用于纯化细胞群获得单细胞的克隆形成试验,国外虽有利用环克隆的方法从脂肪来源细胞中分离培养出分化潜能不同的多种细胞克隆,但未对其进行相关表面抗原的分析,国内也未见有用此种方法进行干细胞研究的报道。本实验拟通过单克隆培养的方法,分离纯化出由单一细胞形成的细胞克隆,达到纯化脂肪组织来源干细胞的目的,并检测其表面抗原的表达,同时针对不同克隆鉴定其成脂分化的潜力,并检测了成脂诱导剂对ADSCs部分相关表面抗原表达的影响,寻找ADSCs成脂分化潜能和表面抗原表达之间的关联。为ADSCs在脂肪组织工程中的应用提供实验依据。目的: 1.提取人体皮下脂肪组织来源干细胞(ADSCs)进行单克隆培养和扩增,以获得单克隆ADSCs,并鉴定不同克隆细胞的干细胞相关表面抗原表达。 2.针对不同克隆ADSCs通过对其生长动力学、形态学、成脂分化能力等方面的特征进行鉴定比较,探索ADSCs成脂分化潜能和表面抗原表达之间的关联,寻找代表定向成脂分化亚群的细胞标志,从而提高构建脂肪组织的成功率。 3.对比成脂诱导前后ADSCs部分相关表面抗原表达的变化,研究成脂诱导剂对ADSCs部分相关表面抗原表达的影响,为ADSCs在脂肪组织工程中的应用奠定实验基础。方法: 临床手术切取脂肪组织,经胶原酶消化法原代培养,细胞培养并传至第2代后,有限稀释法进行克隆形成实验。针对获得的单克隆ADSCs,观察其在体外培养的形态学和生物学特点,并用流式细胞仪检测不同克隆CD29、CD34、CD44、CD54、CD106、ABCG2的表达。各克隆分别进行成脂诱导,油红O染色鉴定分化潜能。第3代的ADSCs细胞分别于成脂诱导7天前、后检测其CD34、CD54、CD106的表达。第4代ADSCs进行成脂、成骨定向诱导分化,油红“O”染色、茜素红染色定性。结果: 从皮下切取的脂肪组织中能培养出大量的ADSCs,通过克隆形成实验共获得10个单克隆细胞群,不同克隆细胞在形态及增殖活力上存在差异,细胞群扩增到第9～11代后可获得细胞数2×10~6,进行后续研究及冻存。干细胞相关标志检测显示:各个克隆群体均高表达CD29(92.9±7.4%)、CD44(94.6±6.8%):低表达ABCG2(2.5±1.4%);但CD34、CD54和CD106的表达在不同群体间有明显差异。不同克隆的成脂分化潜力明显不同,油红“O”染色可见部分含红染颗粒的阳性细胞高达60%,部分为阴性结果,说明ADSCs中存在成脂分化能力不同的细胞群。第3代ADSCs在成脂诱导7天后CD34、CD54和CD106的表达较诱导之前无明显统计学差异。ADSCs成脂诱导分化两周,可见细胞内有大量脂滴,油红“O”染色可见胞浆内有大量红染颗粒;成骨诱导分化两周可肉眼看到白色矿化的颗粒状钙盐沉积,经茜素红染色鉴定可发现成骨细胞及钙盐沉积被红染。结论: 本实验结果证明:皮下脂肪组织经过酶消化原代培养可提取有多向分化能力的干细胞,通过克隆形成试验培养的方法可以纯化ADSCs,获得高纯度具有间充质细胞特性的细胞群,体外多次传代、长期培养并不改变细胞形态和增殖活性。不同的单克隆细胞群之间表面标志的表达和成脂分化能力存在共性和差异。差异较显著的CD34及CD54可能作为ADSCs中高成脂分化细胞群纯化和分选的标志。另外,在成脂分化后CD34、CD54和CD106的表达较之前无显著性差异,与传代培养后的下降变化不同,也提示了这些抗原可能的标识作用。但尚不能认为这些标志物和分化能力之间存在线性关系。后续研究应当对这些标志物进行鉴定,以明确其与成脂分化之间的联系,建立脂肪组织来源基质细胞的纯化技术,为ADSCs构建脂肪组织填充物实现组织工程修复缺损奠定实验基础。
[Abstract]:Background:
Tissue engineering technology, which combines a small amount of seed cells with biomaterials after in vitro amplification, repairs large tissue or organ defects and reconstructs the physiological function of the defect site, provides a new way to solve the problems of tissue defect in clinic, and has attracted wide attention recently.
Adult stem cells can be derived from bone marrow, adipose tissue, skin, muscle and other tissues, including bone marrow stromal cells (BMSCs), which are found early, easy to obtain materials, minimally invasive and the most studied. With the popularization and deepening of research, adipose tissue-derived stem cells (ADSCs) have been gradually recognized by people. Compared with BMSCs, ADSCs are more widely distributed in the body and have more available cells. The advantages of acceptance show great potential in the research and application of tissue engineering.
It is generally believed that ADSCs isolated by common methods are a mixed group of cells with different differentiation potentials and a large number of mixed cells. Only some of them can form adipocytes, which will inevitably affect the success rate of adipose tissue construction. How to improve the purity and purity of stem cells extracted from adipose tissue Finding markers for identifying stem cells has been one of the most pressing problems. Although there have been many reports on the surface antigens of ADSCs, there is still no consensus on the specific surface markers of ADSCs. In addition, many cell clones with different differentiation potentials have been isolated and cultured from adipose-derived cells by cyclic cloning, but no analysis of the related surface antigens has been made. There is no report on the use of this method for stem cell research in China. In order to purify adipose tissue-derived stem cells and detect the expression of their surface antigens, the potential of adipogenic differentiation was identified according to different clones, and the effect of adipogenic inducers on the expression of some surface antigens of ADSCs was detected. To explore the relationship between adipogenic differentiation potential and surface antigen expression of ADSCs and provide experimental basis for the application of ADSCs in adipose tissue engineering.
Objective:
1. Subcutaneous adipose tissue-derived stem cells (ADSCs) were extracted from human adipose tissue for monoclonal culture and amplification to obtain monoclonal ADSCs and to identify the expression of stem cell-related surface antigens in different cloned cells.
2. By identifying and comparing the growth kinetics, morphology and adipogenic differentiation ability of different clones of ADSCs, the relationship between adipogenic differentiation potential and surface antigen expression of ADSCs was explored, and the cell markers representing the adipogenic differentiation subgroup were found to improve the success rate of adipose tissue construction.
3. To compare the expression of ADSCs related surface antigens before and after adipogenic induction, and to study the effect of adipogenic inducers on the expression of ADSCs related surface antigens, so as to lay an experimental foundation for the application of ADSCs in adipose tissue engineering.
Method:
Adipose tissue was harvested and cultured by collagenase digestion. Cells were cultured and passed to the second generation. The morphological and biological characteristics of monoclonal ADSCs were observed in vitro. The expression of CD29, CD34, CD44, CD54, CD106 and ABCG2 were detected by flow cytometry. The expression of CD34, CD54 and CD106 in ADSCs of the 3rd generation was detected before and 7 days after the induction of adipogenesis. The 4th generation ADSCs were adipogenesis, osteogenic differentiation was induced, oil red "O" staining and alizarin red staining were used to characterize.
Result:
A large number of ADSCs can be cultured from subcutaneous adipose tissue. A total of 10 monoclonal cell groups were obtained by clone formation test. The morphological and proliferative activities of different clonal cells were different. The number of ADSCs was 2 CD29 was overexpressed in all clones, CD44 was underexpressed in ABCG2 (2.5+1.4%) and CD34, CD54 and CD106 were significantly different in different populations. The expression of CD34, CD54 and CD106 in ADSCs of the third generation had no significant difference after 7 days of adipogenesis induction compared with that before induction. Alizarin red staining showed that osteoblasts and calcium salt deposits were stained red.
Conclusion:
The results of this experiment showed that the subcutaneous adipose tissue could be digested by enzymatic digestion and cultured to extract multipotent stem cells. ADSCs could be purified by clone formation test. High purity cells with mesenchymal characteristics could be obtained. The cells were subcultured in vitro for many times. Long-term culture did not change the morphology and proliferative activity of the cells. There are similarities and differences in the expression of surface markers and the ability of adipogenic differentiation among the monoclonal cell populations. CD34 and CD54 with significant differences may be used as markers for the purification and sorting of highly adipogenic cell populations in ADSCs. These markers should be identified in order to clarify the relationship between these markers and adipogenic differentiation, establish purification technology of adipose tissue-derived stromal cells, and construct adipose tissue for ADSCs. The experimental basis for filling tissue to repair defects of tissue engineering is established.
【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

【相似文献】