间充质干细胞体外调控脐带血造血前体细胞增殖和分化的研究
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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
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, so
that 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
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
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, so
that 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
引文
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26. Wang ZE. Myles GM. Brandt CS. Lioubin MN. Rohrschneider L. Identification of the ligand-binding regions in the macrophage colony-stimulating factor receptor extracellular domain. Molecular & Cellular Biology. 1993:13(9):5348-59.
27. Coussens L. Van Beveren C, Smith D. Chen E. Mitchell RL. Isacke CM. Verma IM. Ullrich A. Structural alteration of viral homologue of receptor proto-oncogene fms at carboxyl terminus. Nature. 1986;320(6059):277-80.
28. Dormady SP, Bashayan O, Dougherty R, et al. Immortalized multipotential messenchymal cells and the hematopoietic microenvironment. J Hematother Stem Cell Res, 2001;10(1):125-140.
29. Majumdar MK, Thiede MA, Mosca JD, et al.Phenotypic and functional comparison of cultures of
marrow-derived mesenchymal stem cells (MSCs) and stromal cells.J Cell Physiol. 1998:176(1): 57-66.
30. Sherr CJ. Colony-stimulating factor-1 receptor. Blood. 1990;75(1): 1-12.
31. Luo SQ,Zheng DX. Signal transduction mediated by colony stimulating factor-1 receptor. Chi Sci Bull, 1998:43(12):969-974.
32. Odgren PR,Popoff SN, Safadi FF, et al. The toothless osteopetrotic rat has a normal vitamin D binding protein2macrophage activating factor cascade and chondrodysplasia esistant to treatment with colony stimulating factor21 and/or DBF2MAF. Bone, 1999; 25(2): 175-181.
33. Saleem A,Kharbanda S, Yuan ZM et al. Monocyte colony-stimulating factor stimulates binding of phosphatidylinositol 3-kinase to Grb2 sos complexes in human monocytes. J Biol Chem. 1995:270(18):10380-10383.
34. Wang ZE. Myles GM, Brandt CS, et al. Identification of the ligand-binding regions in the macrophage colony-stimulating factor receptor extracellular domain. Mol Cell Biol. 1993;13(9): 5348-5359.
35. Mayani H. Gutiérrez-Rodríguez M, Espinoza L, et al. Kinetics of hematopoiesis in Dexter-type long-term cultures established from human umbilical cord blood cells. Stem Cells 1998:16(2):127-135.
36．邱录贵，Herzig RH．体外扩增脐血CD34+细胞的实验研究．中华血液学杂志．2000：21(8)：417-420
37. Li WM,Huang WQ, Huang YH,et al. Positive and negative haematopoietic cytokines produced by bone marrow endothelial cells. Cytokine, 2000; 12(7): 1017-1023.
38．奚永志，张双喜，孔繁华，等．人骨髓CD34+造血细胞体外扩增形成HPP-CFC造血祖细胞的能力．中国应用生理学杂志，1999：15(1)：66-69．
39. Peter SO, Kittler EL. Ramshaw HS, et al. Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3). IL-6, IL-11. and stem cell factor leads to impaired engraftment in irradiated hosts.. Blood. 1996:87(1):30～37.
40. Varas F, Bernard A, Bueren JA. Restrictions in the stem cell function ofmurine bone marrow grafts after ex vivo expansion of short-term repopulating progenitors. Exp Hematol, 1998;26 (2):100～109.
41. Abkowitz JL, Taboada MR, Sabo KM, Shelton GH. The ex vivo expansion of feline marrow cells leads to increased numbers of BFU-E and CFU-GM but a loss of reconstituting ability. Stem Cells, 1998; 16(4):288～293.
42. Tisdale J F, Hanazono Y, Sellers SE, et al. Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability. Blood, 1998;92(4): 1131～1141.
43. Breems DA, Blokland EAW, Neben S, et al. Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay. Leukemia, 1994;8(7):1095～1104.
44.Thalmeier K, Meissner P, Reisbach G, et al. Establishment of two permanent human bone marrow stromal cell lines with long-term post irradiation feeder capacity Blood, 1994; 83(7):1799～1807.
45.Koller MR, Palsson MA, Manchel I, et al. Long-term culture-initiating cell expansion is dependent on frequent medium exchange combined with stromal and other accessory cell effects. Blood, 1995;86(5): 1784～1793.
46.Koller MR, Manchel I,Brott DA, Passon B. Donor-to-donor variability in the expansion potential of human bone marrow cells is reduced by accessory cells but not by soluble growth factors. Exp Hematol, 1996;24(13):1484～1493.
47.Azizi SA, Stokes D, Augelli BJ, et al.Engraftment and migration of human bone marrow stromal cells im lanted in the brains of albino rats sim ilarities to astrocyte grafts. Proc N atl A cad SciU SA, 1998:95(7):3908-3913.
48.Brandt JE, Galy AH. Luens KM, et al. Bone marrow repopulation by human marrow stem cells after long-term expansion culture on porcine endothelial cell line. Exp Hematol, 1998;26(10):950-961.
49.Deans RJ,Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol. 2000:28(8): 875-884.
50.Koc ON, Gerson SL. Cooper BW, et al. Rapid hematopo ietic recovery after coinfusion of autologous blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol, 2000;18(2): 307-316.
51.Jae-Hong Kim, Seong-Hyun Jeong,Seok-Yun Kang. et al. Co-transplantation of ex vivo culture-expanded human mesenchymal stem cells and allogeneic hernatopoietic stein cell transplantation-report of 12 cases. Blood. 2002;100(1): 183b-184b.
52.Mayani H, Guilbert LJ, Janowska-Wieczorek A. Biology of the hemopoictic microenvironment. Eur J Haematol 1992:49(5):225-233.
53.Metcalf D. Hemopoietic colonies. In vitro cloning of normal and leukemic cells. RecentResults Cancer Res.; 1977:61: 1-227.
54.Piacibello W, Sanavio F, Garetto L, et al. Extensive amplification and selfrenewal of human primitive hematopoietic stem cells from cord blood. Blood 1997;89(8):2644-2653.
55.Gordon MY, Blackett NM. Some factors determining the minimum number of cells required for successful clinical engraftment. Bone Marrow Transplant, 1995,15(5):659-662.
56.Krause DS,Fackler MJ, Civin CI,et al. CD34: Structure,biology,and clinical utility.
Blood, 1996;87(1): 1-13.
57.Brandt J, Briddell RA, Srour EF, ET AL. Role of c-kit ligand in the expansion of human hematopoietic progenitor cells, Blood, 1992;79(3):634-641.
58.Hoffman R, Tong J, Brandt J,et al. The in vitro and in vivo effects of stem cell factor on human hematopoiesis. Stem Cells,1993: 11(2):76-82.
59.Broxmeyer HE, Lu L, Cooper S,et al. Flt3 ligand stimulates/costimulates the growth of myeloid stem/progenitor cells, Exp. Hematol. 1995,23(10). 1121-1129.
60.Lyman SD, Brasel K, Rousseau AM, et al. The flt3 ligand: a hematopoietic stem cell factor whose activities are distinct from steel factor, Stem Cells, 1994(Suppl 1): 12: 99-107 (discussion 108-110).
61.Petzer AL, Zandstra PW, Piret JM,et al. Differential cytokine effects on primitive (CD34 + CD38_) human hematopoietic cells: novel responses to Flt3-ligand and thrombopoietin, J. Exp. Med. 1996;183(6): 2551-2558.
62.Wodnar-Filipowicz A, Lyman SD, Gratwohl A. et al. Flt3 ligand level reflects hematopoietic progenitor cell function in aplastic anemia and chemotherapy-induced bone marrow aplasia, Blood 1996:88(12):4493-4499.
63.Sitnicka E, Lin N, Priestley GV,et al. The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells, Blood, 1996:87(12): 4998-5005.
64.Broudy VC, Lin NL, Kaushansky K. Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro, Blood 1995:85(7): 1719-1726.
65.Young JC. Bruno E, Luens KM, et al. Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+ Thy-1+Lin_ primitive progenitor cells. Blood 1996:88(6): 1619-1631.
66.Kobayashi M, Laver JH, Kato T, et al. Thrombopoietin supports proliferation of human primitive hematopoietic cells in synergy with steel factor and/or interleukin-3, Blood 1996:88(7):429-436.
67.Kishimoto T, Akira S, Narazaki M,et al. Interlcukin-6 family of cytokines and gp130, Blood. 1995;86(4) 1243-1254.
68.Yoshida K, Taga T. Saito M,et al. Targeted disruption of gp130, a common signal transducer for the interleukin 6 family of cytokines, leads to myocardial and hematological disorders, Proc. Natl. Acad. Sci. U. S. A. 1996;93(1): 407-411.
69.Moore KA, Ema H, Lemischka IR, In vitro maintenance of highly purified, transplantable hematopoietic stem cells, Blood 1997,89(12): 4337-4347.
70.Bernad A, Kopf M, Kulbacki R, et al. Interleukin-6 is required in vivo for the regulation of stem cells and committed progenitors of the hematopoietic system, Immunity 1994, 1(9): 725-731.
71.Shah AJ, Smogorzewska EM, Hannum C,et al. Flt3 ligand induces proliferation of quiescent human bone marrow CD34 + CD38_ cells and maintains progenitor cells in vitro, Blood 1996,87(9): 3563-3570.
72.Ikebuchi K, Wong GG, Clark SC, et al. Interleukin 6 enhancement of interleukin 3-dependent proliferation of multipotential hemopoietic progenitors, Proc. Natl. Acad. Sci. U. S. A. 1987,84(24): 9035-9039.
73.Moore MAS. Clinical implications of positive and negative hematopoietic stem Cell regulators. Blood, 1991,78(1): 1-19
74.奚永志，唐佩弦．检测人类原始造血细胞的几种有效途径．中国病理生理杂志，1996，12(12)：442-445．
75.Mayani H. Dragowska W, Lansdorp PM. Characterization of functionally distinct subpopulations of CD34+ cord blood cells in serum-free long-term cultures supplemented with hematopoietic cytokines. Blood 1993:82(9):2664-2672.
76.Tong J. Hoffman R, Siena S et al. Characterization and quantitation of primitive hematopoietic progenitor cells present in peripheral blood antografts. Exp Hematol, 1994(10): 22: 1016-1024.
77.Mayani H. Guilbert LJ, Janowska-Wieczorek A. Modulation of erythropoiesis and myelopoiesis by exogenous erythropoietin in human long-term marrow cultures. Exp Hematol 1990;18(3):174-179.
78.Koury MJ. Programmed cell death (apoptosis) in hematopoiesis. Exp Hematol, 1992: 20(4): 391-394.
2. Majumdar MK,Thiede MA, Mosca JD,et al.Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells.J Cell Physiol 1998;176(1):57-66.
3. Minguell JJ, Erices A, Conger P. M esenchymal stem cells. Exp Biol Med, 2001; 226 (6): 507-520.
4. Friedenstein AJ,Gorskaja JF, Kulagina NN.Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematpl, 1976;4(5):267-274.
5. Azizi SA, Stokes D, Augelli BJ, et al. Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats-similarities to astrocyte grafts. Proc Natl Acad Sct USA. 1998; 95(7):3908-3913.
6. Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum,and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA,1999;96(19):10711-10716.
7. Woodbury D, Schwarz EJ. Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res,2000;61(4):364-370.,
8. Schwartz RE,Reyes M, Koodie L,et al. Multipotent adult progenitor cells from bone marrows differentiate into functional hepatocyte-like cells. J Clin Invest,2002:109(10): 1291-1302.
9. Nuttall ME. Patton AJ,Olivera DL. et al. Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype: implications for osteopenic disorders. J Bone Miner Res, 1998:13(3): 371-382.
10. Fortier LA,Nixon AJ.Williams J, et al. Isolation and chondrocytic differentiation of equine bone marrow-derived mesenchymal stem cells.Am J V et Res, 1998;59(9): 1182-1187.
11. Ghilzon R,McCulloch CA, Zohar R. Stromal mesenchymal progenitor cells in process citation. Leuk Lymphoma, 1999;32(324): 211-221.
12. Zohar R, Sodek J,McCulloch CA. Characterization of stromal progenitor cells enriched by flow cytometry. Blood, 1997;90(9): 3471-3481.
13. Conger PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol, 1999; 181(1):67-73.
14. Minguell JJ, Erices A, Conget P. M esenchymal stem cells.Exp Biol Med, 2001;226(6): 507-520.
15. Piersma AH,Brockbank KG,Ploemacher RE,et al.Characterization of fibroblastic stromal cells from murine
bone marrow.Exp Hematol, 1985;13(4):237-243
16. Owen M.Marrow stromal stem cells.J Cell Sci Suppl, 1988;10(1):63-76.
17. Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 52azacytidine. Muscle Nerve, 1995:18(12):417-428.
18. Pittenger MF,Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999;284(5411):143-147.
19. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999;284(5411): 143-147.
20. Koc ON, Gerson SL, Cooper BW, et al. Rapid hematopoietic recovery after coinfusion of autologous2blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high2dose chemotherapy. J Clin Oncol, 2000; 18(2):307-316.
21. Chaudhary LR, Spelsberg TC, Riggs BL. Production of various cytokines by normal human osteoblast2like cells in response to ILI β and tumor necrosis factor a. Endocrinology, 1997; 130(17):2528-2534.
22. Majumdar MK, Thiede MA, Mosca JD,et al.Phenotypic and functional comparison of cultures of marrowy-derived mesenchymal stem cells (MSCs) and stromal cells. J Cell Physiol. 1998, 176(1): 57-66.
23. Dormady SP, Bashayan O. Dougherty R, et al. Immortalized multipotential messenchymal cells and the hematopoietic microenvironment. J Hematother Stem Cell Res, 2001: 10(1):125-140.
24. Majumdar MK, Thiede MA, Mosca JD. et al. Phenotypic and functional comparison of cultures of marrow2derived mesenchymal stem cells (MSCs) and stromal cells. J Cell Physiol, 1998: 176(1):57-66.
25 Fixe P. Praloran V. M-CSF: haematopoietic growth factor or inflammatory cytokine?Cytokine, 1998: 10(1):32-37.
26. Wang ZE. Myles GM. Brandt CS. Lioubin MN. Rohrschneider L. Identification of the ligand-binding regions in the macrophage colony-stimulating factor receptor extracellular domain. Molecular & Cellular Biology. 1993:13(9):5348-59.
27. Coussens L. Van Beveren C, Smith D. Chen E. Mitchell RL. Isacke CM. Verma IM. Ullrich A. Structural alteration of viral homologue of receptor proto-oncogene fms at carboxyl terminus. Nature. 1986;320(6059):277-80.
28. Dormady SP, Bashayan O, Dougherty R, et al. Immortalized multipotential messenchymal cells and the hematopoietic microenvironment. J Hematother Stem Cell Res, 2001;10(1):125-140.
29. Majumdar MK, Thiede MA, Mosca JD, et al.Phenotypic and functional comparison of cultures of
marrow-derived mesenchymal stem cells (MSCs) and stromal cells.J Cell Physiol. 1998:176(1): 57-66.
30. Sherr CJ. Colony-stimulating factor-1 receptor. Blood. 1990;75(1): 1-12.
31. Luo SQ,Zheng DX. Signal transduction mediated by colony stimulating factor-1 receptor. Chi Sci Bull, 1998:43(12):969-974.
32. Odgren PR,Popoff SN, Safadi FF, et al. The toothless osteopetrotic rat has a normal vitamin D binding protein2macrophage activating factor cascade and chondrodysplasia esistant to treatment with colony stimulating factor21 and/or DBF2MAF. Bone, 1999; 25(2): 175-181.
33. Saleem A,Kharbanda S, Yuan ZM et al. Monocyte colony-stimulating factor stimulates binding of phosphatidylinositol 3-kinase to Grb2 sos complexes in human monocytes. J Biol Chem. 1995:270(18):10380-10383.
34. Wang ZE. Myles GM, Brandt CS, et al. Identification of the ligand-binding regions in the macrophage colony-stimulating factor receptor extracellular domain. Mol Cell Biol. 1993;13(9): 5348-5359.
35. Mayani H. Gutiérrez-Rodríguez M, Espinoza L, et al. Kinetics of hematopoiesis in Dexter-type long-term cultures established from human umbilical cord blood cells. Stem Cells 1998:16(2):127-135.
36．邱录贵，Herzig RH．体外扩增脐血CD34+细胞的实验研究．中华血液学杂志．2000：21(8)：417-420
37. Li WM,Huang WQ, Huang YH,et al. Positive and negative haematopoietic cytokines produced by bone marrow endothelial cells. Cytokine, 2000; 12(7): 1017-1023.
38．奚永志，张双喜，孔繁华，等．人骨髓CD34+造血细胞体外扩增形成HPP-CFC造血祖细胞的能力．中国应用生理学杂志，1999：15(1)：66-69．
39. Peter SO, Kittler EL. Ramshaw HS, et al. Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3). IL-6, IL-11. and stem cell factor leads to impaired engraftment in irradiated hosts.. Blood. 1996:87(1):30～37.
40. Varas F, Bernard A, Bueren JA. Restrictions in the stem cell function ofmurine bone marrow grafts after ex vivo expansion of short-term repopulating progenitors. Exp Hematol, 1998;26 (2):100～109.
41. Abkowitz JL, Taboada MR, Sabo KM, Shelton GH. The ex vivo expansion of feline marrow cells leads to increased numbers of BFU-E and CFU-GM but a loss of reconstituting ability. Stem Cells, 1998; 16(4):288～293.
42. Tisdale J F, Hanazono Y, Sellers SE, et al. Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability. Blood, 1998;92(4): 1131～1141.
43. Breems DA, Blokland EAW, Neben S, et al. Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay. Leukemia, 1994;8(7):1095～1104.
44.Thalmeier K, Meissner P, Reisbach G, et al. Establishment of two permanent human bone marrow stromal cell lines with long-term post irradiation feeder capacity Blood, 1994; 83(7):1799～1807.
45.Koller MR, Palsson MA, Manchel I, et al. Long-term culture-initiating cell expansion is dependent on frequent medium exchange combined with stromal and other accessory cell effects. Blood, 1995;86(5): 1784～1793.
46.Koller MR, Manchel I,Brott DA, Passon B. Donor-to-donor variability in the expansion potential of human bone marrow cells is reduced by accessory cells but not by soluble growth factors. Exp Hematol, 1996;24(13):1484～1493.
47.Azizi SA, Stokes D, Augelli BJ, et al.Engraftment and migration of human bone marrow stromal cells im lanted in the brains of albino rats sim ilarities to astrocyte grafts. Proc N atl A cad SciU SA, 1998:95(7):3908-3913.
48.Brandt JE, Galy AH. Luens KM, et al. Bone marrow repopulation by human marrow stem cells after long-term expansion culture on porcine endothelial cell line. Exp Hematol, 1998;26(10):950-961.
49.Deans RJ,Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol. 2000:28(8): 875-884.
50.Koc ON, Gerson SL. Cooper BW, et al. Rapid hematopo ietic recovery after coinfusion of autologous blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol, 2000;18(2): 307-316.
51.Jae-Hong Kim, Seong-Hyun Jeong,Seok-Yun Kang. et al. Co-transplantation of ex vivo culture-expanded human mesenchymal stem cells and allogeneic hernatopoietic stein cell transplantation-report of 12 cases. Blood. 2002;100(1): 183b-184b.
52.Mayani H, Guilbert LJ, Janowska-Wieczorek A. Biology of the hemopoictic microenvironment. Eur J Haematol 1992:49(5):225-233.
53.Metcalf D. Hemopoietic colonies. In vitro cloning of normal and leukemic cells. RecentResults Cancer Res.; 1977:61: 1-227.
54.Piacibello W, Sanavio F, Garetto L, et al. Extensive amplification and selfrenewal of human primitive hematopoietic stem cells from cord blood. Blood 1997;89(8):2644-2653.
55.Gordon MY, Blackett NM. Some factors determining the minimum number of cells required for successful clinical engraftment. Bone Marrow Transplant, 1995,15(5):659-662.
56.Krause DS,Fackler MJ, Civin CI,et al. CD34: Structure,biology,and clinical utility.
Blood, 1996;87(1): 1-13.
57.Brandt J, Briddell RA, Srour EF, ET AL. Role of c-kit ligand in the expansion of human hematopoietic progenitor cells, Blood, 1992;79(3):634-641.
58.Hoffman R, Tong J, Brandt J,et al. The in vitro and in vivo effects of stem cell factor on human hematopoiesis. Stem Cells,1993: 11(2):76-82.
59.Broxmeyer HE, Lu L, Cooper S,et al. Flt3 ligand stimulates/costimulates the growth of myeloid stem/progenitor cells, Exp. Hematol. 1995,23(10). 1121-1129.
60.Lyman SD, Brasel K, Rousseau AM, et al. The flt3 ligand: a hematopoietic stem cell factor whose activities are distinct from steel factor, Stem Cells, 1994(Suppl 1): 12: 99-107 (discussion 108-110).
61.Petzer AL, Zandstra PW, Piret JM,et al. Differential cytokine effects on primitive (CD34 + CD38_) human hematopoietic cells: novel responses to Flt3-ligand and thrombopoietin, J. Exp. Med. 1996;183(6): 2551-2558.
62.Wodnar-Filipowicz A, Lyman SD, Gratwohl A. et al. Flt3 ligand level reflects hematopoietic progenitor cell function in aplastic anemia and chemotherapy-induced bone marrow aplasia, Blood 1996:88(12):4493-4499.
63.Sitnicka E, Lin N, Priestley GV,et al. The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells, Blood, 1996:87(12): 4998-5005.
64.Broudy VC, Lin NL, Kaushansky K. Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro, Blood 1995:85(7): 1719-1726.
65.Young JC. Bruno E, Luens KM, et al. Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+ Thy-1+Lin_ primitive progenitor cells. Blood 1996:88(6): 1619-1631.
66.Kobayashi M, Laver JH, Kato T, et al. Thrombopoietin supports proliferation of human primitive hematopoietic cells in synergy with steel factor and/or interleukin-3, Blood 1996:88(7):429-436.
67.Kishimoto T, Akira S, Narazaki M,et al. Interlcukin-6 family of cytokines and gp130, Blood. 1995;86(4) 1243-1254.
68.Yoshida K, Taga T. Saito M,et al. Targeted disruption of gp130, a common signal transducer for the interleukin 6 family of cytokines, leads to myocardial and hematological disorders, Proc. Natl. Acad. Sci. U. S. A. 1996;93(1): 407-411.
69.Moore KA, Ema H, Lemischka IR, In vitro maintenance of highly purified, transplantable hematopoietic stem cells, Blood 1997,89(12): 4337-4347.
70.Bernad A, Kopf M, Kulbacki R, et al. Interleukin-6 is required in vivo for the regulation of stem cells and committed progenitors of the hematopoietic system, Immunity 1994, 1(9): 725-731.
71.Shah AJ, Smogorzewska EM, Hannum C,et al. Flt3 ligand induces proliferation of quiescent human bone marrow CD34 + CD38_ cells and maintains progenitor cells in vitro, Blood 1996,87(9): 3563-3570.
72.Ikebuchi K, Wong GG, Clark SC, et al. Interleukin 6 enhancement of interleukin 3-dependent proliferation of multipotential hemopoietic progenitors, Proc. Natl. Acad. Sci. U. S. A. 1987,84(24): 9035-9039.
73.Moore MAS. Clinical implications of positive and negative hematopoietic stem Cell regulators. Blood, 1991,78(1): 1-19
74.奚永志，唐佩弦．检测人类原始造血细胞的几种有效途径．中国病理生理杂志，1996，12(12)：442-445．
75.Mayani H. Dragowska W, Lansdorp PM. Characterization of functionally distinct subpopulations of CD34+ cord blood cells in serum-free long-term cultures supplemented with hematopoietic cytokines. Blood 1993:82(9):2664-2672.
76.Tong J. Hoffman R, Siena S et al. Characterization and quantitation of primitive hematopoietic progenitor cells present in peripheral blood antografts. Exp Hematol, 1994(10): 22: 1016-1024.
77.Mayani H. Guilbert LJ, Janowska-Wieczorek A. Modulation of erythropoiesis and myelopoiesis by exogenous erythropoietin in human long-term marrow cultures. Exp Hematol 1990;18(3):174-179.
78.Koury MJ. Programmed cell death (apoptosis) in hematopoiesis. Exp Hematol, 1992: 20(4): 391-394.