差速消化分离培养脂肪源性内皮祖细胞（EPCs）及其生物学特性的研究

发布时间：2018-03-21 10:37

本文选题：脂肪组织　切入点：内皮祖细胞　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:内皮祖细胞(Endothelial progenitor cells,EPCs)是成熟内皮细胞的前体细胞,可迁移到缺血组织,分化为成熟内皮细胞(Enthothelial cells,ECs),发挥血管新生作用。随着EPCs研究的深入,其在临床诊断、预后判断和各种缺血性疾病的治疗方面将会有广阔的应用前景。EPCs不仅存在于骨髓、外周血和脐血中,还存在于胚胎、心脏、骨骼肌和血管中。然而,这些来源的EPCs都存在一定的限制。因此,寻找合适来源的EPCs就变得尤为重要。近年来的研究发现,脂肪组织中含有多种细胞,包括EPCs。脂肪组织不仅具有来源丰富,获取容易且其对人体创伤较小的优势,而且其种子细胞丰富,适合细胞的自体移植,因此,脂肪组织是理想的EPCs种子细胞来源,但目前国内外缺乏有效的分离培养脂肪源性EPCs的方法。目的:探讨一种有效、经济、可行的从人脂肪组织中分离培养EPCs的方法,并对其生物学特性展开研究。方法:来用酶消化法从人脂肪组织中分离培养出脂肪基质血管细胞(Stromal vascular cells,SVF),流式细胞术检测SVF的免疫表型以分析其细胞成分。通过EPCs和脂肪干细胞(Adipose stem cells,ASCs)对胰蛋白酶的敏感性不同,差速消化分离EPCs和ASCs。观察细胞的形态特征,绘制细胞的生长曲线、计算细胞的细胞倍增数(PD)和倍增时间(DT)评估细胞的生长增殖能力,流式细胞分析术检测EPCs的表型特征,免疫荧光染色观察细胞摄取FITC-UEA-1和吞噬Dil-ac-LDL的能力。最后,通过体外成血管试验分析EPCs的血管形成能力。结果:通过差速消化分离法,我们成功从脂肪组织中分离出EPCs和ASCs。流式细胞术检测分析显示EPCs表达CD31、CD34和VEGFR2,而几乎不表达造血干细胞表面标志CD45;体外扩增培养后呈典型的铺路石样形态,并可吞噬乙酰化低密度脂蛋白(Dil-ac-LDL)和结合荆豆凝集素(FITC-UEA-1),在荧光显微镜下观察吞噬Dil-ac-LDL的EPCs呈红色荧光,而结合FITC-UEA-1呈绿色荧光。此外,将其接种于Matrigel人工基底膜,可形成血管腔样的结构。ASCs高度表达CD29、CD73、CD90、CD105等间充质细胞表面标志,体外扩增培养呈长梭形纤维细胞样生长。结论:我们通过差速消化分离法成功从人脂肪组织中分离培养出EPCs,为脂肪源性EPCs的分离培养提供了一种有效、经济、可行的研究方法,为各种缺血性疾病提供了丰富的种子细胞来源,给治疗性血管新生和再生医学提供了广阔的应用前景。
[Abstract]:Background: Endothelial progenitor cells (EPCs) are progenitor cells of mature endothelial cells, which can migrate to ischemic tissue and differentiate into endothelial cells of endothelial cells (ECs), which play a role in angiogenesis. Prognosis judgment and treatment of various ischemic diseases will have broad application prospects. EPCs exist not only in bone marrow, peripheral blood and umbilical cord blood, but also in embryo, heart, skeletal muscle and blood vessel. EPCs from these sources has certain limitations. Therefore, it is particularly important to find suitable sources of EPCs. Recent studies have found that adipose tissue contains a variety of cells, including EPCs.Adipose tissue is not only rich in sources. Therefore, adipose tissue is an ideal source of EPCs seed cells, because it is easy to obtain and has less trauma to human body, and its seed cells are abundant and suitable for autologous transplantation of cells. But there is a lack of effective method to isolate and culture adipose EPCs at home and abroad. Objective: to explore an effective, economical and feasible method for isolation and culture of EPCs from human adipose tissue. Methods: stromal vascular cells were isolated and cultured from human adipose tissue by enzyme digestion, and the immunophenotype of SVF was detected by flow cytometry. The cell components were analyzed by EPCs. The sensitivity to trypsin is different from adipose stem cells. EPCs and ASCs were separated by differential digestion. The morphological characteristics of cells were observed, cell growth curves were drawn, cell multiplication number and multiplication time were calculated to evaluate cell growth and proliferation ability, and phenotypic characteristics of EPCs were detected by flow cytometry. The ability of FITC-UEA-1 uptake and Dil-ac-LDL phagocytosis was observed by immunofluorescence staining. Finally, the angiogenesis ability of EPCs was analyzed by in vitro vascularization test. We successfully isolated EPCs and ASCs.FCM analysis from adipose tissue showed that EPCs expressed CD31mCD34 and VEGFR2, but hardly expressed CD45. after amplification and culture in vitro, we showed typical paving stone shape. EPCs phagocytosis of Dil-ac-LDL and green fluorescence of FITC-UEA-1 were observed under fluorescence microscope. In addition, it was inoculated into Matrigel artificial basement membrane. ASCs highly expressed CD29, CD73, CD90, CD105 and other mesenchymal cell surface markers. Conclusion: we successfully isolated and cultured EPCs from human adipose tissue by differential digestion, which provides an effective, economical and feasible method for the isolation and culture of adipose derived EPCs. It provides abundant seed cell sources for various ischemic diseases and provides a broad application prospect for therapeutic angiogenesis and regenerative medicine.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2

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