【作者】 郑翠玲；

【导师】 韩忠朝； 陆敏；

【作者基本信息】 中国协和医科大学 ， 内科学， 2009， 博士

【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。更多还原

【Abstract】 Background:Multipotent mesenchymal stem cells(MSC),isolated from many tissues,are multipotent stem cells with the capacity for self-renewal and multilineage differentiation.Their ease of isolation and expansion,their multipotency,their low immunogenicity and immune-modulatory properties present them as an ideal stem cell candidate for tissue engineering and regenerative medicine.An important issue in cellular therapeutics is the availability of alternative MSC sources,while key to the success of cell therapy is the biological properties of MSC.Understanding the biological properties of MSC will be beneficial for fully utilizing the therapeutic potential of MSC,therefore,there has been an increased interest in understanding the biology of MSC due to their potential use in cell-based therapy.To date,most therapeutic applications are based on adult bone marrow(ABM) MSC,but ABM MSC have many drawbacks,including the limited volume,the invasive procedures of their harvest,and the age-dependent decline in the absolute number and differential capacity.This has prompted researchers to look for alternative sources of MSC and suitable culture medium for MSC so as to meet the requirement of clinics and research.Objective:This study aims at identificating the biological properties of MSC cultured with low serum by evaluating their morphous,karyotype, growth kinetics,immunophenotype and differentiation potential during in vitro expansion so as to supply the rationale for their therapeutic potential for tissue engineering.Methods:①Cells were isolated from fetal bone marrow and lung as well as umbilical cord by adherent culture,cultured in the culture medium containing DMEM/DF12 as well as 2%fetal bovine serum,and purified by passaging in vitro.②The karyotype and growth curve of MSC were detected.③Cell cycle was assayed by flow cytometry.④The immunophenotype and the pluripotent marker expression were obtained by flow cytometry analysis.⑤MSC were induced for adipocytes,osteoblasts and chondrocytes in the induction media.Oil red 0,alizarin red and alcian blue staining was performed to examine lipid droplet,mineralization and acid mucopolysaccharide,respectively.The expression of lineage-specific markers such as PPAR-γ,osteocalcin and collagenⅡwas evaluated by flow cytometry analysis.⑥MSC from fetal bone marrow and lung could differentiate into neural cells by two-step introduction and the expression of neural markers including MAP2,Nestin and GFAP in differentiated cells were confirmed by confocal microscopy.⑦The two-step procedure was performed for hepatocyte introduction.The mRNA expression of hepatic specific markers such asαFP,albumin and cytokeratin 18(CK18) was examined by RT-PCR.Hepatocyte function was assessed by detecting glycogen by periodic acid schiff(PAS) staining and dosing albumin in culture supernatants.Results:①MSC cultured with low serum had adherent and spindle-shaped characterizations,grow rapidly,and reached confluence with a whirl pool-like appearance.②They maintained a normal karyotype and displayed the growth curve of ’S’ type.③Most cells were in the G0/G1 phases and only a small proportion of cells actively proliferated as revealed by cell cycles analysis.④They highly expressed mesenchymal markers including CD13, CD29,CD44,CD49e,CD90,CD105,CD166,HLA classⅠ,and did not express hematopoietic and endothelial markers such as CD31,CD34,CD11b,CD45,CD117, CD133 and HLA classⅡ.Importantly,MSC could even express pluripotent markers of embryonic stem cells such as Oct4,Nanog,Sox2 and SSEA-4.The expression levels of all markers in MSC from fetal lung were remained stable through 25 passages.⑤The induced MSC showed positive staining of oil red 0,alizarin red and alcian blue and high expression of lineage-specific markers.The data confirmed their potentials to differentiate directedly into adipocytes,osteoblasts and chondrocytes;moreover,MSC from fetal lung (P25) possessed the similar differentiation potentials.⑥The expression levels of neural markers in the differentiated cells were significantly higher than those in control cells.⑦Compared with control cells,the expression of hepatocyte markers was upregulated in the induced cells that also demonstrated the capability of storing glycogen and secreting albumin.Conclusion:①Culture medium with low serum is good culture reagent for MSC.②MSC cultured in the medium show great proliferation potential and suffice the demand of seed cells for tissue engineering.③They are able to differentiate into adipocytes,osteoblasts and chondrocytes and MSC from fetal lung maintain the ability up to 25 passages,which make it possible to supply enough cells for tissue engineering of bone or cartilage.④MSC express pluripotent markers of embryonic stem cells and differentiat directedly towards extramensenchymal lineages such as neuroectoderm cells and hepatocytes of endoderm.⑤The biological properties of MSC are favourable,and MSC have emerged as excellent seed cells for tissue engineering and regenerative medicine. Background:Multipotent mesenchymal stem cells(MSC) are multipotent stem cells that can differentiate into tissues of mesodermal origin and can be isolated from various tissues.MSC have also been noted to have profound immunomodulatory effects on immune cells,leading to the modulation of several effector functions.Recently,a new CD4+ T-cell subset has been designated as Th17 cells that preferentially produce IL-17.Th17 cells have been widely accepted as important effector cells in the development of autoimmune and allergic diseases and host defenses against a group of pathogens.Whereas the exact mechanisms underlying the immunomodulatory functions of MSC remain largely unknown,these cells have been exploited in a variety of clinical trials aimed at reducing the burden of immune-mediated disease.Objective:This article aimed to investigate the immunoregulatory effects of fetal bone marrow-derived MSC on human T lymphocytes,especially Th17 cells,so as to broaden our understanding of the immunomodulatory properties of MSC and provide insights as to their potential for clinical use as a cell-based therapy for immune-mediated disorders.Methods:We cultured PBMC and CD4+ T cells with or without FBM-MSC,and we named the coculture of PBMC or CD4+ T cells with FBM-MSC as coculture cells.The culture media included PHA and rIL-2,if not specifically mentioned.We used qRT-PCR,ELISA and flow cytometry analyses to compare①the expression levels of IL-17 bewteen PBMC as well as CD4+ T cells and their coculture cells in the absence or presence of PHA and rIL-2;②the change of IL-17 expression when supplemented with additional IL-6,IL-6 neutralizing antibody and TGF-β1;③the expression levels of IL-1 and IL-23 between PBMC as well as CD4+ T cells and their coculture cells;④the expression of IFN-γbetween PBMC as well as CD4+ T cells and their coculture cells;⑤the mRNA expression of Foxp3 between PBMC and its coculture cell.Results:qRT-PCR,ELISA and flow cytometry data showed①that the molecular and cell expression levels of IL-17 in PBMC and CD4+ T cells were lower than those in their coculture cells with or without PHA and rIL-2,respectively(p<0.05);②that the addition of IL-6 increased the expression of IL-17(p<0.05),while the addition of IL-6 neutralizing antibody and TGF-β1 decreased the expression of IL-17(p<0.05);③that the increased concentration of IL-1 in the supernant of CD4+ T cells than that in the supernant of their coculture cells(p<0.05),and that no significant difference of IL-23 expression was found between PBMC as well as CD4+ T cells and their coculture cells(p>0.05);④that the expression levels of IFN-γproduced by Th1 cells in PBMC and CD4+ T cells were lower than those in their coculture cells(p<0.05);⑤that the mRNA expression of Foxp3 in PBMC was lower than that in their coculture cells(p<0.05).Conclusion:Our data demonstrated for the first time that FBM-MSC could positively regulated human Th17 cells.The mechanisms underlying the regulation might include IL-6 as well as IL-1,whereas IL-23 seemed not to involve in the investigated regulation.In addition,FBM-MSC might exert a negative role in IFN-γproducing Th1 cells,but maybe promote Treg.更多还原