本文選題：骨髓 + 間充質(zhì)干細胞　； 參考：《浙江大學》2009年博士論文

【摘要】： 間充質(zhì)干細胞(Mesenchymal Stem Cells,MSCs)是一種具有多向分化潛能的成體干細胞,是當前干細胞研究領(lǐng)域的熱點,在組織器官缺損性疾病、退行性疾病、自身免疫性疾病、遺傳缺陷等疾病的治療中有著巨大的臨床應用前景。隨著MSCs臨床應用的進程,對MSCs生物學特性的研究和認識提出了更高的要求。一直以來,骨髓MSCs被認為是骨髓單個核細胞中具有貼壁的特性、并可在體外培養(yǎng)增殖和誘導多向分化的非造血系統(tǒng)的干細胞。值得注意的是,迄今為止MSCs尚未發(fā)現(xiàn)有特異性的表面分子。目前國際公認的MSCs表型特征為:細胞表面CD73、CD90、CD105等抗原呈陽性,而CD14、CD19、CD34、CD45、HLA-DR等造血譜系分子表達陰性。由于缺乏鑒定細胞的特異性標志,導致對MSCs的生物學特征缺乏嚴格統(tǒng)一的定義,制約了對MSCs生物學特性的研究和認識,使得MSCs的檢測、分離純化以及應用均面臨著巨大的難題。 第1章人間充質(zhì)干細胞特異性單克隆抗體及其抗原特征的鑒定 為發(fā)現(xiàn)新的MSCs特異的表面標志,以期深入研究MSCs的生物學特性,本研究以培養(yǎng)的人骨髓MSCs為免疫原免疫BALB/C小鼠,應用雜交瘤技術(shù)研究制備了具有自主知識產(chǎn)權(quán)的入骨髓MSCs特異性單克隆抗體ZUB1(專利號:ZL200510061034.2),并對ZUB1單克隆抗體及其識別抗原的生物學特性進行了深入研究。通過對抗體種屬和組織細胞特異性鑒定,證實ZUB1單克隆抗體針對人MSCs有高度的特異性和敏感性,ZUB1可識別骨髓、脂肪組織、臍帶組織和臍帶血來源的MSCs,與大鼠、小鼠和兔骨髓MSCs均無交叉反應。Western-blot結(jié)果顯示,ZUB1單克隆抗體識別一種分子量約為250KD的抗原分子,該種抗原在13種人血液系統(tǒng)惡性疾病細胞株和10種人實體組織細胞株均無表達。骨髓、脂肪組織、臍帶組織和臍帶血來源的MSCs在傳代擴增過程中形態(tài)基本一致,流式細胞術(shù)檢測傳代擴增細胞表達ZUB1抗原的陽性率分別為97.58±0.68%、89.50±3.97%、96.63±1.23%、87.23±4.07%,此外CD29、CD44、CD105、CD166、CD146陽性標記出現(xiàn)單峰,CD3、CD14、CD19、CD34、CD117、CD133、CD45、CD235a、HLA-DR均表達陰性,傳代細胞ZUB1抗原和其他各表面抗原表達均無顯著性差異。當誘導骨髓MSCs向成骨細胞和脂肪細胞定向分化時,流式細胞檢測顯示ZUB1抗原表達相應減弱,提示ZUB1抗原可作為MSCs干細胞特征的標志分子,且可能參與了MSCs分化的細胞活動。 為進一步鑒定ZUB1單克隆抗體識別的抗原表位,我們通過Western-blot實驗證實ZUB1單克隆抗體識別的抗原分子量約為250KD,應用ZUB1單克隆抗體通過免疫沉淀的方法從人骨髓MSCs細胞裂解液中獲取與ZUB1抗體特異性結(jié)合的蛋白質(zhì),ZUB1抗體蛋白復合物經(jīng)SDS-PAGE電泳分離后切取蛋白質(zhì)條帶,質(zhì)譜分析提示ZUB1單克隆抗體識別的分子量為250KD的抗原肽段為非肌性肌球蛋白-9重鏈(non-muscle myosin heavy chain 9,NMMHCⅡa)。生物信息學分析提示該類細胞骨架蛋白的同型異構(gòu)體在不同組織和干細胞的不同分化階段有不同的異構(gòu)體的表達,MSCs特異的形態(tài)特征和細胞骨架結(jié)構(gòu)與其多向分化潛能是相適應的。該研究進一步豐富了對骨髓MSCs特異性分子特征的認識,也為進一步研究MSCs分化等細胞活動提供了一個重要的線索。 第2章ZUB1抗原陽性骨髓間充質(zhì)干細胞亞群的鑒定和生物學特性的研究 隨著對體外培養(yǎng)MSCs的生物學特性認識的深入,研究者利用不同的表面分子分離骨髓單個核細胞以期進一步研究和鑒定骨髓原始MSCs及其不同亞群的生物學特性。利用ZUB1單克隆抗體,我們建立了ZUB1~+骨髓MSCs的分離培養(yǎng)體系,并深入研究了ZUB1~+骨髓MSCs亞群的生物學特性。應用ZUB1單克隆抗體通過免疫磁珠分選骨髓單個核細胞,流式細胞儀檢測分選后細胞純度為61.52±6.69%,分選后獲得的ZUB1~+骨髓單個核細胞體積較小、核質(zhì)比大,符合干細胞的形態(tài)學特征。ZUB1~+骨髓單個核細胞培養(yǎng)3天后細胞貼壁呈梭形,5天后細胞擴增明顯,梭形貼壁細胞增殖呈集落樣分布,培養(yǎng)15天左右貼壁細胞達80%～90%匯合,細胞形態(tài)均一,且具有很好的增殖活性,細胞擴增至第8代細胞生長曲線無明顯差異。流式細胞術(shù)分析顯示ZUB1~+骨髓單個核細胞貼壁培養(yǎng)后具有MSCs的表型特性,且可誘導向成骨細胞、脂肪細胞和神經(jīng)元樣細胞。因此,ZUB1單克隆抗體可用于分離富集ZUB1~+骨髓MSCs亞群。 一直以來MSCs缺乏特異性分子標志,目前尚無有效的指標可以鑒定骨髓中不同亞群的原始MSCs的含量。CFU-F可用于評估骨髓單個核細胞中MSCs集落的形成情況,也是目前用于評估骨髓中原始MSCs的數(shù)量的主要指標之一。將未分選的骨髓單個核細胞、及磁珠分選后ZUB1~+和ZUB1~-骨髓單個核細胞按2×10~4/cm~2、1×10~3/cm~2、2×10~4/cm~2的細胞密度分別接種培養(yǎng),細胞培養(yǎng)15d,ZUB1~+骨髓單個核細胞形成CFU-F的數(shù)量和直徑明顯多于未分選的骨髓單個核細胞,而ZUB1~-骨髓單個核細胞未見集落形成。按照每10~5單個核細胞計算ZUB1單克隆抗體分選骨髓單個核細胞的CFU-F富集指數(shù)(Enrichment Factor)為193.09±32.81,且ZUB1~+骨髓MSCs增殖較快。為進一步分析ZUB1~+骨髓MSCs的表型特征,對MSCs公認的一系列表面分子檢測結(jié)果顯示該類細胞的表型一致:CD29、CD105、CD166、CD146表達陽性,而CD3、CD14、CD19、CD33、CD235a、HLA-DR、CD45、CD34、CD133、CD117為陰性。由此,我們認為ZUB1單克隆抗體可有效地富集骨髓單個核細胞中的MSCs,ZUB1抗原可以作為研究骨髓原始MSCs的分子標志;ZUB1~+骨髓MSCs體外培養(yǎng)增殖較快,并可多向分化為成骨細胞、脂肪細胞和神經(jīng)元樣細胞。 本研究制備了具有自主知識產(chǎn)權(quán)的抗入骨髓MSCs特異性單克隆抗體,并證實ZUB1抗原針對人MSCs有高度的特異性,可用于人MSCs的檢測、鑒定以及富集骨髓中原始MSCs。ZUB1抗原為一種在MSCs中特異的細胞骨架蛋白,參與了MSCs定向分化的生命活動。骨髓細胞中ZUB1~+骨髓單個核細胞作為一個新的骨髓MSCs亞群,豐富了對MSCs生物學特性的認識,并為MSCs的研究帶來新契機。
[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.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R329.2

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