人胎盘源间充质干细胞对脐血单个核细胞体外生长的支持作用及体内移植促进造血重建的实验研究

发布时间：2018-09-06 07:20

【摘要】： 间充质干细胞(mesenchymal stem cells,MSCs)是目前倍受关注的一类具有多向分化潜能的成体干细胞。在不同的诱导条件下,MSCs可分化为成骨细胞、软骨细胞、脂肪细胞等。MSCs的主要来源为成人骨髓,但成人骨髓源MSCs的细胞数量及增殖分化潜能会随着年龄的增大而下降,且病毒感染率较高,此外供者MSCs的采集需行骨髓穿刺术,因而MSCs的临床应用存在很大的局限。近年的研究表明,从胎盘中可分离出MSCs,它们和骨髓MSCs一样,在合适的培养条件下具有多向分化能力。 MSCs作为骨髓造血微环境中的重要组成成分,它通过分泌多种造血生长因子以及细胞与细胞间的直接接触等方式作用于造血干细胞(hematopoietic stem cells,HSCs),因而在造血调控中发挥重要的作用。目前,MSCs已被用来体外支持HSCs扩增,体内联合HSCs移植促进造血重建。本研究在成功分离培养人胎盘源间充质干细胞(placenta-derived mesenchymal stem cells,PMSCs)的基础上,探讨人PMSCs在体内和体外支持造血的功能效应。 本文分为两部分。第一部分阐述了人PMSCs的分离培养和鉴定,并探讨人PMSCs对脐血单个核细胞(MNCs)体外生长的支持作用。将人胎盘组织经胶原酶消化、贴壁和传代培养获得人PMSCs,运用流式细胞仪检测其表面标志,分别联合应用β-甘油磷酸钠、维生素C、地塞米松体系和3-异丁基-1-甲基黄嘌呤、胰岛素、吲哚美辛、地塞米松体系将其诱导分化为成骨细胞和脂肪细胞,继而采用碱性磷酸酶检测和vonkossa染色对其进行成骨分化鉴定,采用油红染色对其进行脂肪分化鉴定。将用密度梯度离心法分离的脐血MNCs和传代后的PMSCs进行共培养,通过细胞计数和流式细胞仪分别检测造血细胞的生长情况。结果显示:1.从人胎盘组织中成功分离和培养PMSCs,贴壁细胞呈成纤维细胞状,经流式细胞仪检测其表型为CD29~+、CD44~+、CD105~+、CD106~+、CD166~+、CD34~-、CD45~-、HLA-DR~-;2.PMSCs经成骨诱导3周后,碱性磷酸酶染色呈强阳性,von kossa染色可见明显钙结节。PMSCs经成脂诱导2周后,油红染色呈阳性,有明显的脂滴出现;3.人PMSCs+脐血MNCs共培养组的细胞总数和CD45~+细胞数在各培养时间点均明显高于对照组,在培养第7天,CD45~+、CD14~+及CD19~+细胞数共培养组也明显高于对照组。结果表明,人PMSCs可以作为滋养层细胞有效支持脐血MNCs的体外生长。 第二部分利用同种异基因小鼠骨髓移植模型,比较经骨髓腔内注射(intra-bonemarrow injection,IBMI)和外周静脉输注(intravenous injection,Ⅳ)两种途径移植小鼠骨髓单个核细胞(BMNCs)后受体早期的造血重建情况,建立了小鼠IBMI技术平台,并利用IBMI技术初步探讨了人PMSCs联合脐血MNCs共移植入SCID鼠体内后的早期造血重建效应。以BALB/c小鼠为供体,通过IBMI和Ⅳ两种途径将其BMNCs移植入经致死量辐照预处理的C57BL/6受鼠体内。60只受鼠随机分为3组:骨髓腔内注射高剂量组1(IBM1组)、骨髓腔内注射低剂量组2(IBM2组)、尾静脉注射组(Ⅳ组),每组20只。在骨髓移植后1、3、6、9 d分别计数各组受鼠胫骨骨髓腔内细胞总数,并用流式细胞仪检测供体植入水平(供体来源细胞总数、供体来源髓系细胞数)。以人PMSCs和脐血MNCs为供体,通过IBMI方法或结合Ⅳ方法,将两种细胞联合或单独移植入经亚致死量辐照预处理的SCID受鼠体内。9只受鼠随机分为3组:联合移植A组(PMSCs+脐血MNCs,IBMI)、单独移植B组(脐血MNCs,IBMI)、联合移植C组(PMSCs经IBMI,脐血MNCs经Ⅳ),每组3只。在移植后14 d取各组SCID小鼠注射侧和对侧胫骨腔内骨髓细胞,并用流式细胞仪检测分析人CD34~+、CD45~+细胞的植入水平。结果显示:1.IBM1组和IBM2组注射侧于移植后6 d胫骨骨髓腔内细胞总数、供体来源细胞总数、供体来源髓系细胞总数均明显高于Ⅳ组;2.SCID小鼠移植后14 d,B组注射侧及对侧胫骨骨髓腔内的人CD34~+、CD45~+细胞的百分比均明显低于A组小鼠的注射侧和对侧。结果表明,IBMI较Ⅳ更能促进同种异基因骨髓移植后的早期造血功能重建;人PMSCs可以增加脐血MNCs的植入,促进造血重建。 综上所述,本实验从人胎盘组织中成功分离培养获得PMSCs,人PMSCs在体外对脐血MNCs的生长有明显的支持作用。通过IBMI技术将人PMSCs与脐血MNCs共移植于SCID小鼠体内,人PMSCs可有效促进脐血MNCs的早期造血重建。这为进一步研究人PMSCs体内和体外支持造血的相关分子机理奠定了实验基础。
[Abstract]:At present, mesenchymal stem cells (MSCs) are a kind of adult stem cells with multipotent differentiation potential. Under different induction conditions, MSCs can differentiate into osteoblasts, chondrocytes, adipocytes and so on. The main source of MSCs is adult bone marrow, but the number and proliferation and differentiation potential of MSCs derived from adult bone marrow. In addition, the collection of MSCs from donors requires bone marrow aspiration, so the clinical application of MSCs is limited. Recent studies have shown that MSCs can be isolated from placenta, and they, like bone marrow MSCs, can differentiate into multiple cells under suitable culture conditions.
As an important component of bone marrow hematopoietic microenvironment, MSCs act on hematopoietic stem cells (HSCs) by secreting a variety of hematopoietic growth factors and direct contact between cells. MSCs play an important role in hematopoietic regulation. At present, MSCs have been used to support HSCs amplification in vitro and in vivo. Combined HSCs transplantation promotes hematopoietic reconstitution. In this study, human placenta-derived mesenchymal stem cells (PMSCs) were successfully isolated and cultured to investigate the functional effects of human PMSCs on supporting hematopoiesis in vivo and in vitro.
The first part describes the isolation, culture and identification of human PMSCs, and the supporting effect of human PMSCs on the growth of umbilical cord blood mononuclear cells (MNCs) in vitro. Sodium, vitamin C, dexamethasone system and 3-isobutyl-1-methylxanthine, insulin, indomethacin, dexamethasone system were induced to differentiate into osteoblasts and adipocytes, then alkaline phosphatase detection and vonKossa staining were used to identify the osteogenic differentiation, and oil red staining was used to identify the adipogenic differentiation. The results showed that: 1. PMSCs were successfully isolated and cultured from human placenta tissues. The adherent cells were fibroblast-like, and their phenotypes were CD29~+, CD44~+, CD1 detected by flow cytometry. 05~+, CD106~+, CD166~+, CD34~-, CD45~-, HLA-DR~-; 2. Alkaline phosphatase staining and von Kossa staining were strongly positive in PMSCs after 3 weeks of osteogenesis induction. Oil red staining was positive in PMSCs after 2 weeks of lipogenesis induction, and obvious lipid droplets appeared. 3. The total number of cells and CD45~+ cells in human PMSCs + umbilical cord blood MNCs co-culture group were strongly positive in all cultures. The number of CD45~+, CD14~+ and CD19~+ cells co-cultured on the 7th day was also significantly higher than that of the control group. The results showed that human PMSCs could be used as trophoblast cells to support the growth of umbilical cord blood MNCs in vitro.
In the second part, we compared the early hematopoietic reconstitution of bone marrow mononuclear cells (BMNCs) recipients by intra-bone marrow injection (IBMI) and peripheral intravenous injection (IV) in allogeneic bone marrow transplantation mice, and established a mouse IBMI platform. The early hematopoietic reconstitution effect of human PMSCs combined with umbilical cord blood MNCs co-transplanted into SCID mice was studied by I technique. BMNCs were transplanted into C57BL/6 recipients by IBMI and IV in BALB/c mice. Intramedullary injection of low dose group 2 (IBM2 group), tail vein injection group (IV group), each group of 20. After bone marrow transplantation 1, 3, 6, 9 days were counted the total number of tibial marrow cells in each group, and flow cytometry was used to detect the level of donor implantation (total number of donor-derived cells, number of donor-derived myeloid cells). Human PMSCs and umbilical cord blood MNCs were used as donors, through Nine SCID recipients were randomly divided into three groups: group A (PMSCs + umbilical cord blood MNCs, IBMI), group B (umbilical cord blood MNCs, IBMI), group C (PMSCs via IBMI, umbilical cord blood MNCs via IV), and three in each group. The results showed that: 1. The total number of cells in the tibial bone marrow cavity, the total number of donor-derived cells and the total number of donor-derived myeloid cells in the IBM 1 and IBM 2 groups were significantly higher than those in the IV group 6 days after transplantation. The percentage of CD34~+ and CD45~+ cells in the injected and contralateral tibial bone marrow cavities of D mice was significantly lower than that of A mice 14 days after transplantation.
To sum up, PMSCs were successfully isolated from human placenta and cultured in vitro. Human PMSCs can significantly support the growth of umbilical cord blood MNCs. Human PMSCs and umbilical cord blood MNCs were co-transplanted into SCID mice by IBMI technology. Human PMSCs can effectively promote the early hematopoietic reconstruction of umbilical cord blood MNCs. This is a further study of human PMSCs in vivo and in vitro. In vitro support for hematopoietic related molecular mechanisms laid the experimental foundation.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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