【作者】 王红祥；

【导师】 邹萍；

【作者基本信息】 华中科技大学 ， 血液内科， 2007， 博士

【摘要】 第一部分脂肪基质干细胞与骨髓间充质干细胞生物学特性的比较【目的】分离培养人脂肪基质干细胞,并鉴定其表型,建立脂肪基质干细胞的分离、纯化、扩增和鉴定的方法。比较脂肪基质干细胞(ASC)与骨髓间充质干细胞(MSC)生物学特性上的异同,为ASC的细胞移植治疗提供实验依据。【方法】分离人脂肪组织,胶原酶Ⅰ消化后制备细胞悬液并培养,消化传代以纯化细胞。细胞计数法绘制脂肪基质干细胞的生长曲线,流式细胞术及免疫荧光法检测其表型。密度梯度离心法分离骨髓单个核细胞,有核细胞贴壁法培养获得MSC。对培养前后的ASC和MSC分别进行计数比较,流式细胞仪测定比较ASC和MSC表面抗原表达的异同。通过混合淋巴细胞反应和淋巴细胞转化实验比较这两种细胞免疫学特性的差别。【结果】从脂肪中分离的细胞第3天开始贴壁生长,其倍增时间约为48小时。以后细胞主要呈梭形生长。其表型为CD31- CD34- CD45- HLA-DR- CD29+CD105+ CD13+ CD44+。从骨髓中分离出的有核细胞数多于从脂肪中分离的有核细胞(P<0.001),但培养后获得的干细胞数差别无显著性意义(P>0.05)。ASC和MSC都表达CD13、CD29、CD44、CD105,而在CD49d、CD106的表达上存在差异。相同数量的ASC和MSC在混合淋巴细胞反应、淋巴细胞转化实验中对淋巴细胞的抑制作用相当(P>0.05)。【结论】成功建立了从人脂肪组织中分离培养和扩增基质干细胞的体系。ASC和MSC在细胞表面抗原表达和免疫原性等方面既有相似之处,又存在一些差异,说明ASC是一群不同于MSC的细胞。第二部分脂肪基质干细胞多向分化潜能的研究【目的】研究脂肪基质干细胞(ASC)体外定向分化为心肌样细胞、神经元样细胞、成骨细胞和脂肪细胞的能力,进一步了解ASC的生物学特性。【方法】获取并培养正常人的ASC。5-氮胞苷(5-Aza)诱导分化为心肌样细胞,全反式维甲酸(ATRA)诱导分化为神经元样细胞,不同浓度地塞米松联合维生素C、β-磷酸甘油分别诱导ASC分化为成骨细胞和脂肪细胞。倒置显微镜观察诱导后细胞的形态,逆转录-聚合酶链式反应(RT-PCR)检测心肌相关基因ANP、cTnT、cTnI和αMHC及神经细胞相关基因nestin的表达。免疫荧光法检测cTnT和结蛋白的表达;Western blot法检测connexin43的表达。免疫组织化学染色法和Western blot法检测神经丝蛋白(NF)和神经元烯醇化酶(NSE)的表达。von Kossa染色法检测钙盐沉积,油红O染色法检测诱导后脂肪细胞形态。【结果】ASC经5-氮胞苷的作用可呈现典型的心肌样细胞形态,ANP、cTnT、cTnI和αMHC基因及cTnT、结蛋白和connexin43均呈阳性表达。不同扩增代数的ASC经诱导剂ATRA的作用均呈现典型的神经元样细胞形态,巢蛋白基因及NF、NSE均表达阳性。ASC向成骨细胞诱导后有钙盐沉积,von Kossa染色阳性;向脂肪细胞诱导分化后油红O染色阳性。【结论】ASC具有体外大量扩增并保持低分化状态的特性,以及定向分化为心肌样细胞、神经元样细胞、成骨细胞和脂肪细胞的能力,是一种可用于组织工程的理想的种子细胞。第三部分脂肪基质干细胞对异基因T淋巴细胞和树突状细胞的影响及机制探讨【目的】研究ASC对异基因T淋巴细胞和树突状细胞表型及分泌细胞因子的影响,探讨ASC发挥免疫调节作用的方式和可能机制。【方法】(1)将ASC上清和ASC分别与异基因T淋巴细胞进行混合培养。MTT法检测T细胞的增殖,AnnexinⅤ法检测凋亡,流式细胞术检测T淋巴细胞中CD4+CD25+细胞的比例,ELISA法检测T细胞分泌IL-10和TGF-β1的水平,RT-PCR法检测FOXP3基因的表达。(2)将ASC上清和ASC分别与异基因树突状细胞(DC)进行混合培养。流式细胞术检测CD80、CD83和CD86的表达,ELISA法检测DC表达IL-12的水平,RT-PCR法检测吲哚胺2,3双加氧酶(IDO)基因的表达。【结果】(1)ASC上清对T淋巴细胞的增殖和凋亡无明显影响。ASC对T淋巴细胞的生长有抑制作用,但对其凋亡无影响。ASC上清和ASC均能增加CD4+CD25+T细胞在T淋巴细胞中的比例,增加T细胞分泌IL-10和TGF-β1的水平,上调FOXP3基因的表达。(2)ASC上清与DC共培养后,使DC表达CD80、CD83和CD86减少;ASC上清与ASC均能降低DC分泌IL-12的水平,并上调IDO基因的表达。【结论】ASC能通过细胞直接接触和分泌细胞因子这两种方式分别作用于T淋巴细胞和树突状细胞,发挥免疫负调节作用,并参与诱导机体的免疫耐受。更多还原

【Abstract】 PARTⅠComparison Study of Biologic Character of Human Adipose Stromal Cells and Bone Marrow Mesenchymal Stem Cells【Objective】This study aimed to compare the biologic character of adipose stromal cells (ASC) and bone marrow mesenchymal stem cells (MSC), construct a series of methods of isolation, purification, proliferation and identification of ASC, and make a foundation for cell therapy based on ASC.【Methods】Human adipose tissues were isolated, and digested with typeⅠcollagenase solution. Then the cells were cultured in plastic culture flasks. The numbers of cells were counted and cell growth curve were drawn. Flow cytometry and immunofluorescence were used to identify their phenotype. At the same time, mononuclear cells were isolated from human bone marrow and MSC were derived by cultivation. The numbers of ASC and MSC were counted before and after the cultivation. The surface phenotype of the two populations of cells was examined by flow cytometry. Effects of ASC or MSC on mixed lymphcyte response and PHA-induced lymphocyte transformation were investigated.【Results】The cells isolated from human adipose tissue displayed a fibroblast-like morphology adhering to the culturing plate 3 days later, and double counts time was 48 hours. The phenotype of ASC was CD31- CD34- CD45- HLA-DR- CD29+ CD105+ CD13+ CD44+. The numbers of nucleated cells isolated from bone marrow was significantly higher than those from adipose tissue (P<0.001), while no significant differences were observed for the yield of adherent stromal cells (P>0.05). Both cells expressed CD13、CD29、CD44、CD105. Differences in expression were noted for CD49d and CD106. The same number of ASC and MSC showed comparatively negative immunomodulative functions by inhibiting the mixed lymphocyte response and induction of transformation (P>0.05).【Conclusion】Adipose stromal cells were successfully cultured. ASC and MSC showed both similarities and differences in the features of cell surface markers and immunosuppressive properties. So ASC was some kind of cells quite different from MSC.PARTⅡMultiple Differentiative Potent of Adipose Stromal Cells【Objective】This study aimed to observe the capacity of human adipose stromal cells (ASC) to differentiate into cardiomyocyte-like cells, neuron-like cells, osteoblasts and adipocytes in vitro.【Methods】ASC was isolated and cultured, then induced by 5-Azacytidine (5-aza), all-trans retinoic acid (ATRA), various concentrations of dexamethasone combined with vitamine C andβ-glycerophosphate, respectively. Microscope was used to observe their morphology. Reverse-transcription polymerase chain reaction was used to detect the expression of ANP, cTnT, cTnI,αMHC and nestin. Immunofluorescence and western blot were used to detect the expression of cTnT, desmin and connexin43, respectively. Immunohistochemistry and Western blot were used to detect the expression of neurofilament (NF) and neuron specific enolase (NSE). Von Kossa staining was used to detect the deposit of calcium salts and Oil O staining to detect the leipo-drops.【Results】ASC expressed ANP, cTnT, cTnI,αMHC, desmin and connexin43 after adding 5-Aza in the culture system. Various passages of ASC all have the capacity of neuron differentiation. They expressed nestin, NF and NSE 10 days after adding ATRA in the culture system. ASC can also differentiate into osteoblasts and adipocytes under suitable conditions.【Conclusion】ASC has the character of proliferation and low-differentiation. Under suitable conditions, they can differentiate into cardiomyocyte-like cells, neuron-like cells, osteoblasts and adipocytes in vitro. ASC can be used as an alternative source of cells for tissue engineering.PARTⅢEffects of Adipose Stromal Cell on Allo-genetic T lymphocytes and Dendritic Cells and Its Possible Role【Objective】This study aimed to observe the effect of adipose stromal cells (ASC) on the phenotype and secreting cytokines of allogenetic T lymphocytes and dendritic cells (DC), and tried to elucidate the possible role of the immunomodulation of adipose stromal cell.【Methods】(1) The supernatant of ASC and ASC itself were co-cultured with allogenetic T lymphocytes, respectively. MTT assays were used to detect the proliferative rates of T cells. Annexin-Ⅴ/PI staining was used to detect the apoptotic rates, and flow cytometry to detect the proportion of CD4+CD25+ cells. Enzyme-linked immunoabsorbent assay was used to detect the secretion level of IL-10 and TGF-β1. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression level of FOXP3 gene.(2) The supernatant of ASC and ASC itself were co-cultured with allogenetic dendritic cells. Flow cytometry was used to detect the expression of CD80, CD83 and CD86. Enzyme-linked immunosorbent assay was used to detect the secretion level of IL-12, RT-PCR to detect the expression level of indoleamine 2, 3-dioxyenase (IDO).【Results】(1) The supernatant of ASC had no deep impact on the proliferation and apoptosis of T lymphocytes. ASC had the direct inhibitory effect on the growth of T lymphocytes, while did not affect the apoptosis. Both the supernatant of ASC and ASC itself can increase the proportion of CD4+CD25+ cells in T lymphocytes, and increase the level of IL-10 and TGF-β1. They can also up-regulate the expression level of FOXP3 gene.(2) The expression level of CD80, CD83 and CD86 on the surface of dendritic cells was down-regulated after the supernatant of ASC were added on the culture system of DC. The secretion of IL-12 was reduced, while the expression of IDO was up-regulated in the presence of ASC supernatant and ASC.【Conclusion】ASC can affect the function of T lymphocytes and DC via both cell-to-cell contact and secretion cytokines. Through these ways ASC showed a kind of negative immunomodulative effect and played an important role on inducing immune tolerance.更多还原