本文選題：間充質(zhì)干細(xì)胞　切入點(diǎn)：骨髓微環(huán)境　出處：《中國(guó)人民解放軍醫(yī)學(xué)院》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：間充質(zhì)干細(xì)胞（Mesenchymal stem cells，MSCs）是骨髓中除造血干細(xì)胞外另一重要的干細(xì)胞。MSCs具有自我更新和多向分化能力的特點(diǎn)，它可以分化為多種骨髓基質(zhì)細(xì)胞如脂肪細(xì)胞、成骨細(xì)胞和內(nèi)皮細(xì)胞等；在一定的誘導(dǎo)體系中還能被誘導(dǎo)分化為軟骨細(xì)胞、肌細(xì)胞、心肌細(xì)胞和神經(jīng)元等。因此，MSCs對(duì)于受損組織的修復(fù)具有重要意義。另外MSCs還具有免疫調(diào)控和支持造血的能力，而且來(lái)源廣泛，容易在體外培養(yǎng)和擴(kuò)增，正是因?yàn)檫@些特點(diǎn)，MSCs目前已經(jīng)應(yīng)用于臨床并顯示了良好的治療效果。但是，許多關(guān)鍵問題如免疫調(diào)控的具體機(jī)制、骨髓微環(huán)境對(duì)MSCs特性的影響等問題并未解決，所以，建立一種方便、快捷、有效地體外分離培養(yǎng)擴(kuò)增MSCs的方法能為后續(xù)的研究奠定良好的基礎(chǔ)。 本實(shí)驗(yàn)應(yīng)用一種新的方法——骨髓加骨片法分離培養(yǎng)小鼠MSCs。通過(guò)將骨髓加骨片法與現(xiàn)有的全骨髓貼壁法和骨片消化法對(duì)比，發(fā)現(xiàn)這種新方法出現(xiàn)的MSCs集落時(shí)間最早，集落數(shù)最多，獲得的細(xì)胞數(shù)也最多；細(xì)胞呈梭形貼壁生長(zhǎng)，流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示細(xì)胞高表達(dá)干細(xì)胞標(biāo)志之一Sca-1，高表達(dá)CD44、CD29，不表達(dá)白細(xì)胞表面抗原CD45和內(nèi)皮細(xì)胞標(biāo)志CD31；得到的MSCs在成骨誘導(dǎo)體系和成脂誘導(dǎo)體系中能分別向成骨細(xì)胞和脂肪細(xì)胞分化，具有典型的MSCs特性。 由于MSCs上述多種優(yōu)越的特性，尤其是對(duì)許多組織的損傷修復(fù)能力，使得MSCs各方面功能的研究成為熱點(diǎn)，，而MSCs又是骨髓微環(huán)境中的一種重要的干細(xì)胞，雖然它對(duì)骨髓微環(huán)境中其它細(xì)胞有重要作用如支持造血等，但其本身特性也會(huì)受到骨髓微環(huán)境的影響，當(dāng)機(jī)體年老時(shí)，損傷修復(fù)過(guò)程中MSCs的動(dòng)員和遷移就比年輕機(jī)體要慢，所以本研究著重探討年輕骨髓和年老骨髓對(duì)MSCs遷移和增殖的影響。 通過(guò)年輕骨髓和年老骨髓與骨片共培養(yǎng)，發(fā)現(xiàn)年老骨髓有抑制MSCs集落形成的作用。為了分析骨髓細(xì)胞中何種成分發(fā)揮作用，流式細(xì)胞術(shù)檢測(cè)年輕骨髓和年老骨髓細(xì)胞結(jié)果顯示B220+細(xì)胞在年老骨髓中所占比例明顯低于在年輕骨髓中的比例，而CD11b+、CD3+、Gr-1+和F4/80+細(xì)胞則是在年老骨髓中所占比例明顯高于在年輕骨髓中的比例。用磁珠分選年老骨髓中CD11b+、B220+、Ter119+細(xì)胞并分別與骨片共培養(yǎng)，發(fā)現(xiàn)這三個(gè)亞群并不能抑制MSCs集落形成。在增殖實(shí)驗(yàn)中，年老骨髓也并沒有起到明顯的抑制細(xì)胞增殖的作用，但是在劃痕損傷修復(fù)實(shí)驗(yàn)和遷移實(shí)驗(yàn)中，年老骨髓作用的MSCs的遷移能力要弱于年輕骨髓作用的MSCs，進(jìn)一步的QPCR結(jié)果顯示年輕骨髓刺激后的MSCs表達(dá)SDF-1水平顯著高于年老骨髓刺激后。另外western blot結(jié)果顯示在年輕骨髓或是年老骨髓刺激下，MSCs的MAPK通路均被激活，但是p-38和ERK的磷酸化水平基本沒有差異，而JNK磷酸化水平則是年輕骨髓刺激后的MSCs表達(dá)高。 綜上所述，骨髓加骨片法是一種方便、快捷、有效的小鼠骨髓MSCs分離方法；年老骨髓微環(huán)境具有抑制MSCs從干細(xì)胞龕中遷出的作用，但是具體是骨髓中哪部分細(xì)胞、細(xì)胞外基質(zhì)或是細(xì)胞因子起作用還有待進(jìn)一步研究。
[Abstract]:Mesenchymal stem cells (Mesenchymal stem cells, MSCs) bone marrow mesenchymal stem cells and other important.MSCs stem cell self-renewal and multilineage differentiation capability, it can differentiate into a variety of bone marrow stromal cells such as adipocytes, osteoblasts and endothelial cells; in certain induction system can were induced to differentiate into cartilage cells, muscle cells, myocardial cells and neurons. Therefore, MSCs has an important significance for the repair of damaged tissue. MSCs also has the ability to support hematopoiesis and immune regulation, and a wide range of sources, easy amplification in vitro, it is because of these characteristics, MSCs has been used in clinical application and showed good therapeutic effect. However, the specific mechanisms such as immune regulation of many key problems, the problem of bone marrow microenvironment influence on the character of MSCs has not been resolved, therefore, to build a convenient, fast Czechoslovakia, effective isolation and culture of MSCs in vitro, can lay a good foundation for subsequent research.
The experimental application of a new method, bone marrow and bone slices were isolated and cultured in MSCs. mice by bone marrow and bone graft method and whole bone marrow adherent method and the existing bone digestion method comparison, found the new method of MSCs colony in the earliest time, colony number, the most number of cells obtained; the cells were spindle shaped adherent growth, flow cytometry showed that the expression of stem cell markers of Sca-1, high expression of CD44, CD29, the expression of leukocyte surface antigen CD45 and endothelial cell marker CD31; the MSCs in osteogenic induction system and adipogenic induction system respectively into osteoblasts and fat cell differentiation, MSCs has typical characteristics.
Because of the variety of MSCs superior characteristics, especially the damage repair capacity for many organizations, the study of MSCs function in all aspects has become a hot spot, and MSCs is a kind of important stem cells in bone marrow microenvironment, although there are other cells of the bone marrow microenvironment plays an important role as supporting hematopoiesis, but itself the characteristics will be affected by the bone marrow microenvironment, when the body is old, mobilization and MSCs damage repair process in migration than the young body to slow, so this study focuses on the influence of young and old bone marrow on the migration and proliferation of MSCs.
The young and old bone marrow and bone marrow were cultured, found that elderly bone marrow suppression of MSCs colony formation. In order to analyze what elements play a role in bone marrow cells, flow cytometry in young and old bone marrow cells showed that B220+ cells accounted for in old bone marrow ratio was significantly lower in the proportion of young bone marrow CD11b+, CD3+, Gr-1+, and F4/80+ cells are occupied in the old bone marrow was significantly higher than in young bone marrow ratio. Using CD11b+, B220+ multisort old bone marrow, Ter119+ cells were co cultured with bone slices, found that these three subgroups did not inhibit MSCs colony formation in proliferation. In the experiment, the old bone marrow also did not play a significant role in the inhibition of cell proliferation, but the scratch damage repair assay and migration experiment, the migration ability of old bone marrow effect of MSCs is much weaker than the young bone Curative effects of MSCs, further QPCR results showed that young bone marrow stimulated MSCs SDF-1 expression level was significantly higher than that of old bone marrow stimulation. In addition Western blot results showed that in young or old bone marrow bone marrow stimulation, MAPK MSCs pathway is activated, but the phosphorylation level of P-38 and ERK basically no difference, and JNK phosphate the level is high expression of young bone marrow stimulation after MSCs.
In summary, bone marrow and bone slices method is a convenient, fast and effective method for separating MSCs old mouse bone marrow; bone marrow microenvironment can inhibit MSCs migration from the stem cell niche in the role, but which is a specific part of bone marrow cells, extracellular matrix and cytokines play a role remains to be further studied.

【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R782

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 羅琳,劉文勵(lì);骨髓微環(huán)境造血調(diào)控的研究進(jìn)展[J];國(guó)外醫(yī)學(xué).輸血及血液學(xué)分冊(cè);2004年02期

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