【作者】 岑洪；

【导师】 林茂芳；

【作者基本信息】 浙江大学 ， 内科学， 2004， 博士

【摘要】 骨髓组织中至少含有两种类型的干细胞：造血干细胞(hematopoietic stem cell，HSC)和间充质干细胞(mesenchymal stem cell，MSC)。骨髓造血微环境为HSC提供生长、发育的场所。造血微环境细胞成分主要由骨髓MSC分化产生的成纤维细胞、脂肪细胞、成骨细胞和内皮细胞等基质细胞构成。基质细胞通过与造血细胞直接接触、分泌细胞外基质及多种细胞因子调控造血，维持造血微环境的结构和功能完整性，实现对造血的精细调控，维持骨髓正常造血。MSC作为造血微环境主要细胞成分的前体细胞(Precursor)，具有自我更新和多向分化特性，在造血微环境和造血调控中发挥重要作用。近年来有报道，MSC与HSC联合移植能够促进HSC的植活率，缩短无髓期，减少死亡率。但目前对MSC与造血调控之间的关系所知甚少。本研究的目的，是探讨MSC是否能在体外调控造血，以及如何调控造血。本研究分为三个部分：一、脐带血造血前体细胞在间充质干细胞微环境中向单核系分化。二、可溶性M-CSF受体(sMR)的克隆、原核表达和功能测定。三、脐带血造血前体细胞在间充质干细胞微环境中的增殖动力学研究。 第一部分的研究中，采用Ficoll密度梯度离心，得到低密度的骨髓单个核细胞，然后利用MSC的粘附特性来分离纯化MSC。实验中观察到原代培养接种的细胞于3～4h后开始贴壁，24h后贴壁细胞数量明显增多，3～4d出现一些集簇，此时，细胞多为梭形。一周后，贴壁细胞体积增大，大部分呈梭形，有些细胞呈三角形或多角形，两周后，细胞铺成单层，排列成漩涡状、网状、辐射状，其形态与成纤维细胞相似。在原代培养中，还观察到一些圆形的造血细胞或内皮细胞贴壁生长，但经过传代培养后，这些细胞不能再次贴壁，贴壁的均为梭形细胞，因此MSC得到纯化。利用流式细胞术检测体外培养的MSC纯度，结果显示，MSC均表达CD29(99.7％)、CD44(99.1％)和CD166(83.2％)，不表达CD34、CD45和HLA-DR，表明培养的细胞为均一的MSC，无造血细胞污染。为证实体外培养的细胞是MSC，须证明其具有多向分化潜能。本研究通过添加成骨培养基和成脂肪培养基，诱导MSC向成骨细胞和脂肪细胞分化。MSC经体外诱导培养三周后，通过RT-PCR技术检测，证明有成骨特异性基因浙江大学博士学位论文 (骨桥蛋白基因)和成脂肪特异性基因(脂蛋白脂肪酶基因)的表达;通过Von Kossa染色，观察到成骨诱导培养后，细胞外基质有大量钙盐沉积，培养四至六周后油红染色，观察到成脂肪诱导培养后，细胞内有大量的脂肪。表明MSC已向成骨细胞和脂肪细胞分化，证实体外培养的细胞是具有多向分化潜能的Msc。 为观察MSC能否在体外调控造血，将脐带血单个核细胞接种到以MSC为饲养层细胞的培养体系中，经过2一3周的共培养，观察到有大量的造血细胞粘附在MSC上生长。流式细胞术检测，发现该类细胞几乎都表达单核系标志CD14(98.4%)，而不表达其它髓系和淋巴系标志:CD15(0.35%，粒系)、CD41(0.91%，巨核系)、glyeoporinA(O，28%，红系)、CD7 (l .15%，T淋巴细胞)和CD19(0 .91%，B淋巴细胞)。说明在本研究的培养条件下，以MSC为饲养层细胞的微环境中，即使不添加外源性造血生长因子，脐带血造血前体细胞能向单核系定向分化。国内外未见有类似的研究报道。此外，还观察到在向单核系分化的过程中，造血细胞粘附在MSC上生长，如果没有MSC饲养层细胞，只用MSC上清液培养时，细胞会逐渐死亡。提示在向单核系定向分化过程中，除了造血生长因子的作用外，细胞与细胞间的相互作用可能也是必须的。 已知与单核系定向分化有关的细胞因子有GM一cSF和M一cSF。为探讨脐带血造血前体细胞在MSC微环境中向单核系定向分化的机制，检测MSC中造血生长因子的表达，发现其能构成性表达SCI子、I了1 t3L和M一CSF，不表达GM一CSF不[IG一CSF。巨噬细胞集落刺激因子(maCroph:，g。CO王ony一Stim。lating factor，M一CS内是一种谱系特异性的造血生长因子，对单核一巨噬细胞的增殖、分化及活性维持有重要作用。M一CSF与巨噬细胞集落刺激因子受体(M一CSF一附结合后，M一CSF一R发生二聚体化，其胞内区的激酶结构域活化，引发一系列的信号传递，从而发挥其生物学效应。M一CSF一R是原癌基因(fms)编码的跨膜蛋白，属于酪氨酸激酶受体家族，其胞外区有5个19样结构域分别称为D1、D2、D3、D4和D5，现已确定M一CSF一尺胞外区5个19样结构域中的前3个Dl一3对M一C舒的结合起直接作用。 为研究MSC微环境中M一CSF对脐带血造血前体细胞增殖和分化的影响，第二部分的实验克隆了M一CSI了一R胞外配体结合区(Dl一3)(可溶性M一CSF受体，sMR)，通过它封闭间充质干细胞分泌的M一CSF，观察其生物学效应。参考文献报道的M一CSF一R基因序列，设计了一对引物，在上游引物中加入了限制性内切酶Ndel的酶切位点，在下游引物中加入了Ba翩I的酶切位点和终止密码子，扩增SMR的基因序列，然后利用TA克隆的方法，将其连接到pUCm一T载体上。通过测序，证实了克隆的DNA序列完全正确。再利用 Nde工和BamHI限制性内切酶分别切割携带有目的基因的pUCm--T/SMR质粒和表达载体更多还原

【Abstract】 It is known that there are two kind of stem cell in bone marrow, one is hematopoietic stem cell (HSC) and the other is Mesenchymal stem cell(MSC). MSC has been proved to have multilineage differentiation potential and can differentiate into hematopoietic stromal cells, adipocytes,fibroblasts,and osteogenic precursor cells,which are comprised of hematopoietic microenvironment. They provide cell-to-cell interactions, expression and presentation of hematopoietic growth factors,and the secretion of extracellular matrix proteins. Bone marrow microenvironment provide a favorable platform for the localization, self-renewal,and differentiation of hematopoietic stem cell. But now the relation between MSC and HSC remain to be unknown. In this study, we try to explore if MSC can regulate hematopoiesis in vitro and what effects it has.In part I research, a bone marrow aspirate was collected and processed using density gradient centrifugation, from which light-density cells were taken and plated in a DMEM media containing 10%FBS. After allowing 1 day for adherence to culture flask, nonadherent cells were removed. After a 14-day primary expansion period, MSC nearly reached confluency, and these cells had a fibroblast-like morphology. Confluent cultures could be processed further by trypsinization and expansion through sequential passages to confluency. MSC at passage 3 were analysed by flow cytometry, MSC were uniformly positive for CD29(99.7%),CD44(99.1%) and CD166(83.2%), negative for CD34,CD45 and HLA-DR. the result verified that there were not hematopoietic cells in these cells isolated and cultured in vitro.There were not any exclusive Immunophenotypic markers which had been found in MSC, sothat it is impossible to identify it directly. The only way to do it is to assess its multipotent differentiation ability. The differentiation ability of MSCs was assessed in our research. Osteogenic differentiation was assessed by incubating the cells with DMEM containing 10% FBS supplemented with 10-8 M dexamethasone, 0.2 mM ascorbic acid, and 10 mM glycerol phosphate, adipogenic differentiation was assessed by incubation with DMEM containing 10% FBS supplemented with 10-7 M dexamethasone, 10-9 M insulin. RT-PCR assay showed that osteoblast and adipocyte expressed osteopontin and LpL respectively after induced culture., cytochemical staining results displayed that osteogenic differentiation was positive for Von Kossa staining and adipogenic differentiation was positive for Oil-red-O. staining. These data also implied that the cells isolated and cultured in vitro were MSC.To investigate hematopoietic regulation ability of MSC in vitro, MSC was used as feeder cells, but they had never been irradiated, because after irradiation, these cells might possibly undergo morphologic , phenotypic , and regulatory changes that make them unpredictable surrogates for their normal cell counterparts. Mononuclear cells (MNC) isolated from umbilical cord blood cells were seeded in 6-well plates in which MSC had been seeded and had reached confluence. Control experiments were also performed by :1) culturing cord blood MNC in the absence of a feeder cell layer, 2) culturing cord blood MNC supplemented with the supernatant of MSCs in the absence of a feeder cell layer .The cocultures were incubated at 37 in 5% CO2 in air in DMEM-LG medium supplemented with 10% FBS.Under this culture condition, MSC can survive and FBS was so poor that the effect of unknown factor in FBS might be minimized. There were few adherent hematopoietic cells could be observed during the first week,most cells were nonadherent and even the number of hematopoietic cells were reduced. After 10 days of coculture, many cells started to attach tightly to MSC and proliferated rapidly over the feeder cells.Some colonies could be seen in the early stage,but later it was difficult to recognize them because of the rapid proliferation of hematopoietic cells which made the colonies overlapped.After 21 days of coculture, the medium was removed and cells were washed twice with PB更多还原