低血清浓度培养的间充质干细胞特性及其对人Th细胞的免疫调节作用

发布时间：2018-07-02 11:44

本文选题：低血清培养基 + 间充质干细胞　；参考：《中国协和医科大学》2009年博士论文

【摘要】： 背景:间充质干细胞(multipotent mesenchymal stem cells,MSC)是一种存在于多种组织中的具有自我更新和多向分化能力的多能干细胞。MSC具有易于分离和扩增、多向分化、低免疫原性和免疫调节的特性,这些特性使它们成为组织工程和再生医学的理想种子细胞。细胞治疗中的一个重要问题是MSC替代来源的的可获得性,而细胞治疗的成功的决定性因素则是MSC的生物学特性。对MSC生物学特性的理解有利于充分的利用MSC的治疗潜能,因此,MSC生物学特性的研究越来越引起人们的兴趣。到目前为止,大多数的治疗性应用是以成体骨髓来源的MSC为研究对象,但是成体骨髓MSC存在骨髓体积有限,获取标本时需进行侵袭性操作以及MSC的绝对数量和分化能力会随着年龄增加而下降等不足。这就需要研究人员寻找替代的MSC细胞来源,同时,研制良好的细胞培养试剂为MSC的生长和扩增提供适合的环境,从而满足临床治疗和研究方面的需求。目的:本研究旨在鉴定低血清浓度完全培养基培养的MSC的一般特性如形态、核型、增殖动力学、表型特点和分化潜能等生物学性状,从而提供一种MSC的培养试剂并为MSC作为组织工程的种子细胞提供理论基础。方法:①应用贴壁培养的方法从胎儿的骨髓和肺组织以及脐带中分离获得单个细胞,在低血清完全培养基中培养,除去非贴壁的细胞、通过体外传代培养使细胞纯化。②测定培养的MSC的核型和生长曲线。③应用流式细胞仪对培养的MSC进行细胞周期分析。④运用流式细胞技术检测MSC的免疫表型和胚胎干细胞的全能标志物。⑤在诱导体系中定向诱导MSC向脂肪细胞、成骨细胞和软骨细胞分化,利用油红0、茜素红和阿尔新蓝染色观测脂滴形成、钙盐沉积和酸性粘多糖合成情况;利用流式细胞仪检测系分化特异性标志物过氧化物酶体增生物激活受体-γ(PPAR-γ)、骨钙蛋白(osteocalcin)和Ⅱ型胶原的表达。⑥采用两步诱导方案使骨髓和肺组织来源的MSC体外诱导分化为神经细胞,利用激光共聚焦显微镜检测诱导细胞中的神经细胞标志物微管相关蛋白2(microtubule- associated protein2,MAP2)、Nestin和神经胶质酸性蛋白(glial fibrillary acidic protein,GFAP)的表达。⑦采用两步诱导方法使肺组织来源的MSC体外诱导分化为肝细胞,利用RT-PCR方法检测诱导细胞中的肝细胞标志物如甲胎蛋白(αFP)、白蛋白(albumin)和细胞角蛋白18(cytokeratin18,CK18)的表达;利用糖原染色和培养上清中白蛋白的分泌水平对诱导后细胞进行功能性检测。结果:①低血清浓度完全培养基培养的MSC贴壁生长和呈长梭形,细胞生长速度快,细胞融合时出现漩涡状外观。②MSC具有正常核型以及典型的“S”型生长曲线。③细胞周期表明大多数细胞处于G0/G1期,少部分的细胞处于活跃增殖期。④MSC高表达间充质细胞标志物如CD13,CD29,CD44,CD90,CD49e,CD105,CD166和HLA-Ⅰ,而不表达造血细胞和内皮细胞的标志物,如CD31、CD34、CD11b、CD45、CD117,CD133和HLA-Ⅱ。更重要的是,表达胚胎干细胞标志物Oct4,Nanog,Sox2和SSEA-4,其中传代到第25代(P25)的肺组织来源的MSC仍可保持免疫表型和胚胎干细胞标志物表达的稳定。⑤MSC经诱导后,油红0、茜素红和阿尔新蓝染色均为阳性,且高表达系分化特异性标志物,说明其能够分化为脂肪细胞、骨细胞和软骨细胞,其中肺组织来源的MSC能保持其三系分化能力到P25。⑥神经细胞标志物在诱导细胞中的表达水平显著高于其在对照细胞中的水平。⑦与对照细胞相比,肝细胞标志物在诱导细胞中的表达上调;诱导后细胞具有储存糖原和分泌白蛋白的功能。结论:①低血清浓度完全培养基是一种很好的培养MSC的细胞试剂。②利用该培养基培养的MSC具有较强增殖能力,可满足组织工程种子细胞的需要。③利用该培养基培养的MSC可在体外定向分化为脂肪组织、骨组织和软骨组织,而且肺组织来源的MSC能够将这种多系分化潜能保持到P25,从而为进一步构建组织工程化骨或软骨提供了充足的细胞来源。④利用该培养基培养的MSC表达胚胎干细胞的标志物,而且能在体外分化为中胚层以外的细胞如外胚层的神经细胞和/或内胚层的肝细胞。⑤利用该培养基培养的MSC具有良好的生物学特性,可作为组织工程和再生医学的优秀种子细胞。背景:间充质干细胞(multipotent mesenchymal stem cells,MSC)是能分化为中胚层组织的多能干细胞,它可以从机体的各种组织中分离得到。MSC还因其对免疫细胞的免疫调节作用而备受关注,该调节作用能调节多种免疫效应功能。最近,一个新的CD4+T细胞亚类被命名为Th17细胞,它特异性地优先表达IL-17。Th17细胞被认为是自身免疫性疾病和变态反应性疾病发展过程和宿主对抗病原体过程中的重要效应细胞。虽然MSC免疫调节功能的准确机制还不是很清楚,但是MSC已被用于多种临床试验,旨在降低免疫介导的疾病的负担。目的:研究胎儿骨髓间充质干细胞(FBM-MSC)对人T细胞的免疫调节作用。方法:我们在存在或不存在FBM-MSC的情况下培养PBMC和CD4+T细胞,PBMC或CD4+T细胞与FBM-MSC的共培养被称为共培养细胞。除非特别说明,培养基均包含PHA和rIL-2。我们采用实时定量PCR,ELISA和流式细胞分析等方法检测:①正常人外周血单个核细胞(PBMC)和CD4+T细胞及其与FBM-MSC共培养中IL17的表达水平,观察FBM-MSC对Th17细胞的调节作用;②添加外源性的IL-6,IL-6中和抗体,转化生长因子-β1(TGF-β1)后IL17表达水平的改变,观察IL-6通路在FBM-MSC调节IL17中的作用;③正常人PBMC和CD4+T细胞及其与FBM-MSC共培养中IL-1和IL-23在FBM-MSC调节IL17中的作用;④PBMC和CD4+T细胞及其与FBM-MSC共培养中IFN-γ的表达水平,研究FBM-MSC对Th1细胞的调节作用;⑤PBMC及其与FBM-MSC共培养中Foxp3的表达水平,初步研究FBM-MSC对Treg的调节作用。结果:实时定量PCR,ELISA和流式细胞分析等结果表明:①在含有或不含有PHA和rIL-2刺激的情况下,IL-17在PBMC和CD4+T细胞中的分子和细胞表达水平均显著低于其在共培养中的水平(p＜0.05);②添加外源性的IL-6能增加IL-17的表达水平,而添加外源性的IL-6中和抗体则会显著的降低IL-17的表达水平,添加外源性的TGFβ1会导致IL-17表达水平的降低;③IL-1在CD4+T细胞培养上清中的浓度低于其在共培养中的浓度(p＜0.05),而IL-23在PBMC中的转录水平与其在共培养中的水平无明显差异(p＞0.05),IL-23在CD4+T细胞中的蛋白分泌水平与其在共培养中的水平亦无明显差异(p＞0.05);④Th1细胞产生的IFN-γ在PBMC和CD4+T细胞中的mRNA和蛋白表达水平显著低于其在共培养中的水平(p＜0.05);⑤Foxp3在PBMC中的mRNA表达水平低于其在共培养中的水平(p＜0.05)。结论:FBM-MSC对人Th17细胞具有正性调节作用。IL-6是该调节作用的机制之一,IL-1亦可能是其中的一个机制,而IL-23似乎在我们研究的调节作用中不发挥作用。另外,FBM-MSC对人Th1细胞具有抑制作用并且能抑制Th1细胞产生IFN-γ,而对Treg细胞具有促进作用。
[Abstract]:Background: multipotent mesenchymal stem cells (MSC) is a kind of pluripotent stem cell with self renewal and multidirectional differentiation in a variety of tissues..MSC has the characteristics of easy separation and amplification, multidifferentiation, low immunogenicity and immune regulation. These properties make them a tissue engineering and regenerative medicine. One of the important problems in cell therapy is the availability of MSC alternative sources, and the decisive factor in the success of the cell therapy is the biological characteristics of MSC. The understanding of the biological characteristics of MSC is beneficial to the full utilization of the therapeutic potential of MSC. Therefore, the study of the biological characteristics of MSC is becoming more and more popular. So far, most of the therapeutic applications are based on the MSC of adult bone marrow origin, but adult bone marrow MSC has a limited volume of bone marrow, the need for invasive operations to obtain specimens and the decline of the absolute number and differentiation ability of MSC as the age increases. This requires researchers to find alternative MSC. Cell source, meanwhile, develop good cell culture reagents to provide suitable environment for MSC growth and expansion, so as to meet the needs of clinical treatment and research.
Objective: the purpose of this study was to identify the general characteristics of MSC with low serum concentration, such as morphology, karyotype, proliferation kinetics, phenotypic characteristics and differentiation potential, so as to provide a MSC culture reagent and provide a theoretical basis for MSC as a seed cell of tissue engineering.
Methods: (1) single cells were isolated from the bone marrow, lung tissue and umbilical cord of the fetus by using the method of adherent culture. The cells were cultured in the low serum complete medium, removed non adherent cells and purified by external generation and culture. (2) the karyotype and growth curve of the cultured MSC were measured. (3) the flow cytometry was applied to the culture of the cultured MSC. Cell cycle analysis. (4) using flow cytometry to detect the immunophenotype of MSC and the omnipotent marker of embryonic stem cells. (5) induced MSC to differentiate into adipocytes, osteoblasts and chondrocytes in the induction system, and use oil red 0, alizarin red and alcian blue staining to observe lipid droplets, calcium salt deposition and acid mucopolysaccharide synthesis. The expression of peroxisome activation receptor - gamma (PPAR- gamma), osteocalcin (osteocalcin) and type II collagen were detected by flow cytometry (FCM). 6. The two step induction scheme was used to induce the bone marrow and lung tissue derived MSC into neural cells in vitro, and the laser confocal microscopy was used to detect the induced refinement. The expression of microtubule related protein 2 (microtubule- associated protein2, MAP2), Nestin and glial acidic protein (glial fibrillary acidic protein, GFAP) in the cell. (7) the two step induction method was used to induce the lung tissue source to differentiate into liver cells in vitro, and the hepatocytes were detected by the RT-PCR method. The expression of cell markers such as alpha fetoprotein (alpha FP), albumin (albumin) and cytokeratin 18 (cytokeratin18, CK18), and the functional detection of the induced cells by glycogen staining and the secretion of albumin in the supernatant.
Results: (1) the MSC adherent growth and long spindle shape in the low serum concentration culture medium, the cell growth speed and the whirlpool appearance in the cell fusion. (2) MSC has normal karyotype and the typical "S" type growth curve. (3) cell cycle indicates that most cells are in G0/G1 stage, and few cells are in active proliferation period. (4) MSC High expression of mesenchymal cell markers such as CD13, CD29, CD44, CD90, CD49e, CD105, CD166 and HLA- I, but not the markers of hematopoietic and endothelial cells, such as CD31, CD34, CD11b, CD45, etc. MSC can still maintain the stability of the immunophenotype and the expression of the markers of embryonic stem cells. 5. After induction, the staining of oil red 0, alizarin red and Alicia blue were both positive, and the high expression line was differentiated to differentiate into adipocytes, bone cells and chondrocytes, and the MSC of the lung tissue derived from the lung tissue could maintain its differentiation. The expression level of the nerve cell markers in the induced cells was significantly higher than that in the control cells. The expression of hepatocyte markers was up regulated in the induced cells compared with the control cells, and the cells had the function of storing glycogen and secreting albumin compared with the control cells.
Conclusion: (1) the full medium of low serum concentration is a good cell reagent for the cultivation of MSC. (2) the MSC has strong proliferation ability and can meet the needs of tissue engineering seed cells. (3) the MSC can be differentiated into adipose tissue, bone tissue and cartilage tissue, and lung tissue in vitro. The source of MSC can maintain this multilineage differentiation potential to P25, thus providing a sufficient cell source for further construction of tissue engineered bone or cartilage. (4) using the culture medium MSC to express the markers of embryonic stem cells and to differentiate into neurons and / or endoderm cells outside the mesoderm in vitro, such as the ectoderm and / or the inner embryo. The MSC cultured in this medium has good biological characteristics and can be used as an excellent seed cell for tissue engineering and regenerative medicine.
Background: multipotent mesenchymal stem cells (MSC) is a pluripotent stem cell capable of differentiating into mesoderm tissue. It can be isolated from various tissues of the body and can be isolated from the body..MSC is also concerned because of its immunomodulatory effect on immune cells. This regulation can regulate a variety of immune effects. Recently, a new CD4+ is used. The subclass of T cells is named Th17 cell, and its specific priority expression of IL-17.Th17 cells is considered to be an important effector cell in the process of autoimmune and allergic disease and the host against pathogens. Although the exact mechanism of MSC immunoregulation is not clear, MSC has been used in a variety of clinical trials. The experiment was designed to reduce the burden of immune mediated diseases.
Objective: To study the immunomodulatory effect of fetal bone marrow mesenchymal stem cells (FBM-MSC) on human T cells.
Methods: PBMC and CD4+T cells were cultured in the presence or absence of FBM-MSC. Co culture of PBMC or CD4+T cells with FBM-MSC was called co culture cells. Unless specifically explained, the medium contained PHA and rIL-2.. We used real-time quantitative PCR, ELISA, and flow cytometry to detect: (1) normal human peripheral blood mononuclear cells ( PBMC) and CD4+T cells and the expression level of IL17 in co culture with FBM-MSC, and observe the regulatory effect of FBM-MSC on Th17 cells; (2) adding exogenous IL-6, IL-6 neutralizing antibodies, and transforming growth factor beta 1 (TGF- beta 1) to the expression level of IL17, and observing the role of IL-6 pathway in FBM-MSC regulation. The role of IL-1 and IL-23 in FBM-MSC regulation of IL17 in co culture with FBM-MSC; (4) PBMC and CD4+T cells and the expression level of IFN- gamma in co culture with FBM-MSC, and to study the regulation of FBM-MSC on Th1 cells.
Results: the results of real-time quantitative PCR, ELISA and flow cytometry showed that: (1) the expression level of IL-17 in PBMC and CD4+T cells was significantly lower than that in co culture (P < 0.05) in both PBMC and CD4+T cells with or without PHA and rIL-2 stimulation, and the addition of exogenous IL-6 could increase the expression level of IL-17. The IL-6 neutralization antibody of the source significantly reduced the expression level of IL-17, and the addition of exogenous TGF beta 1 could lead to the decrease of IL-17 expression level; (3) the concentration of IL-1 in the culture supernatant of CD4+T cells was lower than that in co culture (P < 0.05), and the level of IL-23 in PBMC was not significantly different from that in co culture (P >) 0.05) the protein secreting level of IL-23 in CD4+T cells was not significantly different from that in co culture (P > 0.05); (4) the level of mRNA and protein expression of IFN- gamma produced by Th1 cells in PBMC and CD4+T cells was significantly lower than that in co culture (P < 0.05); (5) Foxp3 in PBMC was lower than in co culture. The level (P < 0.05).
Conclusion: FBM-MSC has positive regulatory effect on human Th17 cells, and.IL-6 is one of the mechanisms of this regulation. IL-1 may also be one of the mechanisms, and IL-23 seems to not play a role in our study. In addition, FBM-MSC has a inhibitory effect on human Th1 cells and can inhibit the production of IFN- gamma in Th1 cells, and to Treg cells. It has a promoting effect.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392;R329.2

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