一个新的人间充质干细胞特异性表面抗原的鉴定和间充质干细胞亚群生物学特性的研究

发布时间：2018-05-09 08:02

  本文选题：骨髓 + 间充质干细胞　； 参考：《浙江大学》2009年博士论文

【摘要】： 间充质干细胞(Mesenchymal Stem Cells,MSCs)是一种具有多向分化潜能的成体干细胞,是当前干细胞研究领域的热点,在组织器官缺损性疾病、退行性疾病、自身免疫性疾病、遗传缺陷等疾病的治疗中有着巨大的临床应用前景。随着MSCs临床应用的进程,对MSCs生物学特性的研究和认识提出了更高的要求。一直以来,骨髓MSCs被认为是骨髓单个核细胞中具有贴壁的特性、并可在体外培养增殖和诱导多向分化的非造血系统的干细胞。值得注意的是,迄今为止MSCs尚未发现有特异性的表面分子。目前国际公认的MSCs表型特征为:细胞表面CD73、CD90、CD105等抗原呈阳性,而CD14、CD19、CD34、CD45、HLA-DR等造血谱系分子表达阴性。由于缺乏鉴定细胞的特异性标志,导致对MSCs的生物学特征缺乏严格统一的定义,制约了对MSCs生物学特性的研究和认识,使得MSCs的检测、分离纯化以及应用均面临着巨大的难题。 第1章人间充质干细胞特异性单克隆抗体及其抗原特征的鉴定 为发现新的MSCs特异的表面标志,以期深入研究MSCs的生物学特性,本研究以培养的人骨髓MSCs为免疫原免疫BALB/C小鼠,应用杂交瘤技术研究制备了具有自主知识产权的入骨髓MSCs特异性单克隆抗体ZUB1(专利号:ZL200510061034.2),并对ZUB1单克隆抗体及其识别抗原的生物学特性进行了深入研究。通过对抗体种属和组织细胞特异性鉴定,证实ZUB1单克隆抗体针对人MSCs有高度的特异性和敏感性,ZUB1可识别骨髓、脂肪组织、脐带组织和脐带血来源的MSCs,与大鼠、小鼠和兔骨髓MSCs均无交叉反应。Western-blot结果显示,ZUB1单克隆抗体识别一种分子量约为250KD的抗原分子,该种抗原在13种人血液系统恶性疾病细胞株和10种人实体组织细胞株均无表达。骨髓、脂肪组织、脐带组织和脐带血来源的MSCs在传代扩增过程中形态基本一致,流式细胞术检测传代扩增细胞表达ZUB1抗原的阳性率分别为97.58±0.68%、89.50±3.97%、96.63±1.23%、87.23±4.07%,此外CD29、CD44、CD105、CD166、CD146阳性标记出现单峰,CD3、CD14、CD19、CD34、CD117、CD133、CD45、CD235a、HLA-DR均表达阴性,传代细胞ZUB1抗原和其他各表面抗原表达均无显著性差异。当诱导骨髓MSCs向成骨细胞和脂肪细胞定向分化时,流式细胞检测显示ZUB1抗原表达相应减弱,提示ZUB1抗原可作为MSCs干细胞特征的标志分子,且可能参与了MSCs分化的细胞活动。 为进一步鉴定ZUB1单克隆抗体识别的抗原表位,我们通过Western-blot实验证实ZUB1单克隆抗体识别的抗原分子量约为250KD,应用ZUB1单克隆抗体通过免疫沉淀的方法从人骨髓MSCs细胞裂解液中获取与ZUB1抗体特异性结合的蛋白质,ZUB1抗体蛋白复合物经SDS-PAGE电泳分离后切取蛋白质条带,质谱分析提示ZUB1单克隆抗体识别的分子量为250KD的抗原肽段为非肌性肌球蛋白-9重链(non-muscle myosin heavy chain 9,NMMHCⅡa)。生物信息学分析提示该类细胞骨架蛋白的同型异构体在不同组织和干细胞的不同分化阶段有不同的异构体的表达,MSCs特异的形态特征和细胞骨架结构与其多向分化潜能是相适应的。该研究进一步丰富了对骨髓MSCs特异性分子特征的认识,也为进一步研究MSCs分化等细胞活动提供了一个重要的线索。 第2章ZUB1抗原阳性骨髓间充质干细胞亚群的鉴定和生物学特性的研究 随着对体外培养MSCs的生物学特性认识的深入,研究者利用不同的表面分子分离骨髓单个核细胞以期进一步研究和鉴定骨髓原始MSCs及其不同亚群的生物学特性。利用ZUB1单克隆抗体,我们建立了ZUB1~+骨髓MSCs的分离培养体系,并深入研究了ZUB1~+骨髓MSCs亚群的生物学特性。应用ZUB1单克隆抗体通过免疫磁珠分选骨髓单个核细胞,流式细胞仪检测分选后细胞纯度为61.52±6.69%,分选后获得的ZUB1~+骨髓单个核细胞体积较小、核质比大,符合干细胞的形态学特征。ZUB1~+骨髓单个核细胞培养3天后细胞贴壁呈梭形,5天后细胞扩增明显,梭形贴壁细胞增殖呈集落样分布,培养15天左右贴壁细胞达80%～90%汇合,细胞形态均一,且具有很好的增殖活性,细胞扩增至第8代细胞生长曲线无明显差异。流式细胞术分析显示ZUB1~+骨髓单个核细胞贴壁培养后具有MSCs的表型特性,且可诱导向成骨细胞、脂肪细胞和神经元样细胞。因此,ZUB1单克隆抗体可用于分离富集ZUB1~+骨髓MSCs亚群。 一直以来MSCs缺乏特异性分子标志,目前尚无有效的指标可以鉴定骨髓中不同亚群的原始MSCs的含量。CFU-F可用于评估骨髓单个核细胞中MSCs集落的形成情况,也是目前用于评估骨髓中原始MSCs的数量的主要指标之一。将未分选的骨髓单个核细胞、及磁珠分选后ZUB1~+和ZUB1~-骨髓单个核细胞按2×10~4/cm~2、1×10~3/cm~2、2×10~4/cm~2的细胞密度分别接种培养,细胞培养15d,ZUB1~+骨髓单个核细胞形成CFU-F的数量和直径明显多于未分选的骨髓单个核细胞,而ZUB1~-骨髓单个核细胞未见集落形成。按照每10~5单个核细胞计算ZUB1单克隆抗体分选骨髓单个核细胞的CFU-F富集指数(Enrichment Factor)为193.09±32.81,且ZUB1~+骨髓MSCs增殖较快。为进一步分析ZUB1~+骨髓MSCs的表型特征,对MSCs公认的一系列表面分子检测结果显示该类细胞的表型一致:CD29、CD105、CD166、CD146表达阳性,而CD3、CD14、CD19、CD33、CD235a、HLA-DR、CD45、CD34、CD133、CD117为阴性。由此,我们认为ZUB1单克隆抗体可有效地富集骨髓单个核细胞中的MSCs,ZUB1抗原可以作为研究骨髓原始MSCs的分子标志;ZUB1~+骨髓MSCs体外培养增殖较快,并可多向分化为成骨细胞、脂肪细胞和神经元样细胞。 本研究制备了具有自主知识产权的抗入骨髓MSCs特异性单克隆抗体,并证实ZUB1抗原针对人MSCs有高度的特异性,可用于人MSCs的检测、鉴定以及富集骨髓中原始MSCs。ZUB1抗原为一种在MSCs中特异的细胞骨架蛋白,参与了MSCs定向分化的生命活动。骨髓细胞中ZUB1~+骨髓单个核细胞作为一个新的骨髓MSCs亚群,丰富了对MSCs生物学特性的认识,并为MSCs的研究带来新契机。
[Abstract]:Mesenchymal Stem Cells (MSCs) is a kind of adult stem cells with multiple differentiation potential. It is a hot spot in the field of stem cell research. It has a great clinical application in the treatment of tissue and organ defects, degenerative diseases, autoimmune diseases, genetic defects and other diseases. With the clinical application of MSCs The process has raised higher requirements for the study and understanding of the biological characteristics of MSCs. Bone marrow MSCs has been considered to be a adherent characteristic in bone marrow mononuclear cells and can be cultured in vitro to proliferate and induce multidirectional differentiation of non hematopoietic stem cells. It is worth noting that so far MSCs has not been found to be specific. Surface molecules. The internationally recognized MSCs phenotypes are characterized by positive CD73, CD90, CD105 and other antigens on the surface of the cells, while CD14, CD19, CD34, CD45, and HLA-DR are negative. The lack of a specific marker for identification of cells leads to a lack of a strict and unified definition of the biological characteristics of MSCs and restricts the biological characteristics of MSCs. Research and understanding make the detection, separation, purification and application of MSCs face enormous challenges.
The first chapter is the identification of human mesenchymal stem cell specific monoclonal antibodies and their antigenic characteristics.
In order to discover new MSCs specific surface markers, in order to study the biological characteristics of MSCs, this study uses the cultured human bone marrow MSCs as immunogenic immunogenic BALB/C mice. The specific monoclonal antibody ZUB1 (patent number: ZL200510061034.2), which has independent intellectual property right, is prepared by hybridoma technology (patent number: ZL200510061034.2) and to ZUB1 McAb. The biological characteristics of antibodies and their identification antigens have been studied. The specificity and sensitivity of ZUB1 monoclonal antibodies against human MSCs are confirmed by identification of antibody species and tissue cell specificity. ZUB1 can identify bone marrow, adipose tissue, umbilical cord tissue and MSCs from umbilical cord blood, and no MSCs in rats, mice and rabbit's bone marrow MSCs. The cross reaction.Western-blot results showed that ZUB1 monoclonal antibody identified a molecular weight of about 250KD, which had no expression in 13 human hematological malignancies and 10 human body tissue cells. Bone marrow, adipose tissue, umbilical cord tissue and MSCs from umbilical cord blood were formed in the process of passage amplification. The positive rates of ZUB1 antigen expressed by flow cytometry were 97.58 + 0.68%, 89.50 + 3.97%, 96.63 + 1.23% and 87.23 + 4.07% respectively. In addition, CD29, CD44, CD105, CD166, CD146 positive markers appeared single peak, CD3, CD14, CD19, CD34, CD117, CD133, and other surfaces. There was no significant difference in the expression of antigen. When the osteoblast and adipocytes were induced to differentiate into bone marrow MSCs, the flow cytometry showed that the expression of ZUB1 antigen was weakened accordingly, suggesting that ZUB1 antigen could be used as a marker of the characteristics of MSCs stem cells, and may be involved in the activity of MSCs differentiated cells.
In order to further identify the antigen epitopes identified by ZUB1 monoclonal antibody, we confirmed that the molecular weight of the ZUB1 monoclonal antibody was about 250KD by the Western-blot experiment, and the ZUB1 monoclonal antibody was used to obtain the protein of the specific binding of the ZUB1 antibody from the human bone marrow MSCs cell lysate through the immunoprecipitation method, and the ZUB1 antibody protein was obtained. Protein bands were removed by SDS-PAGE electrophoresis. Mass spectrometric analysis suggested that the antigen peptide segment identified by ZUB1 monoclonal antibody was non myoosin -9 heavy chain (non-muscle myosin heavy chain 9, NMMHC II a). Bioinformatics analysis suggested that the homoisomers of this kind of fine cytoskeleton protein were in different tissues and in different tissues. There are different isomers in different stages of stem cell differentiation, and the specific morphological characteristics and cytoskeleton structure of MSCs are adaptable to their multidirectional differentiation potential. This study further enriches the understanding of the specific molecular characteristics of bone marrow MSCs, and provides an important clue to further study the cell activity of MSCs differentiation.
The second chapter is about the identification and biological characteristics of ZUB1 antigen positive bone marrow mesenchymal stem cells subsets.
With the deep understanding of the biological characteristics of MSCs in vitro, the researchers used different surface molecules to separate bone marrow mononuclear cells in order to further study and identify the biological characteristics of bone marrow primitive MSCs and its different subgroups. Using ZUB1 monoclonal antibody, we established the separation and culture system of ZUB1~ + bone marrow MSCs, and studied in depth. The biological characteristics of ZUB1~+ bone marrow MSCs subgroup were detected by using ZUB1 monoclonal antibody to separate bone marrow mononuclear cells by immunomagnetic beads. The purity of the cells was 61.52 + 6.69% after the flow cytometry. The size of the mononuclear cells of the ZUB1~+ bone marrow was smaller, the nucleation ratio was larger, and the morphological characteristics of the confluent stem cells were.ZUB1~+ bone marrow mononuclear cells. After 3 days of cell culture, cell adherence was spindle shaped, and the cell proliferation was obvious in 5 days. The proliferation of spindle adherent cells was distributed in a colony like distribution, and the adherent cells were confluence of 80% to 90% in the culture of 15 days. The cell morphology was uniform, and the cell proliferation activity was good. The cell expansion to the eighth generation cell growth curve had no obvious difference. Flow cytometry analysis showed ZUB1~+ Bone marrow mononuclear cells have the phenotypic characteristics of MSCs after adherent culture, and can induce osteoblasts, adipocytes and neuron like cells. Therefore, ZUB1 monoclonal antibodies can be used to separate and enrich the MSCs subgroup of ZUB1~+ bone marrow.
There has been a lack of specific molecular markers in MSCs. There is no effective indicator to identify the original MSCs content of different subgroups in bone marrow,.CFU-F can be used to evaluate the formation of MSCs colony in bone marrow mononuclear cells, and is one of the main indicators used to evaluate the number of primitive MSCs in bone marrow. ZUB1~+ and ZUB1~- bone marrow mononuclear cells were inoculated at the cell density of 2 x 10~4/cm~2,1 x 10~3/cm~2,2 x 10~4/cm~2 after the separation of nuclear cells and magnetic beads. The cells culture 15d. The number and diameter of CFU-F in ZUB1~+ bone marrow mononuclear cells were obviously more than that of undivided mononuclear cells, but there was no colony in ZUB1~- bone marrow mononuclear cells. The CFU-F enrichment index (Enrichment Factor) of bone marrow mononuclear cells (Enrichment Factor) was 193.09 + 32.81 for each mononuclear cell of each mononuclear cell (McAb), and the proliferation of ZUB1~+ bone marrow MSCs was faster. In order to further analyze the phenotypic characteristics of ZUB1~+ bone marrow MSCs, the recognized list surface molecular detection results of MSCs showed that the form of this kind of cell was displayed. CD29, CD105, CD166, CD146 are positive, and CD3, CD14, CD19, CD33, CD235a, HLA-DR, CD45, CD34, and negative. It can be differentiated into osteoblasts, adipocytes and neuron like cells.
This study has prepared a specific monoclonal antibody against bone marrow MSCs with independent intellectual property rights, and confirmed that ZUB1 antigen is highly specific to human MSCs. It can be used for the detection of human MSCs, identification and enrichment of primitive MSCs.ZUB1 antigen in bone marrow as a specific cytoskeleton in MSCs, and participates in the life of MSCs directed differentiation. ZUB1~+ bone marrow mononuclear cells in bone marrow cells, as a new subgroup of bone marrow MSCs, enrich the understanding of the biological characteristics of MSCs and bring new opportunities for the study of MSCs.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329.2

【共引文献】

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