本文選題：骨髓間充質(zhì)干細胞 + 培養(yǎng)��； 參考：《浙江大學》2005年碩士論文

【摘要】：骨髓中除含有能分化發(fā)育成各種血細胞的造血干細胞之外,還含有可產(chǎn)生非造血 組織的間充質(zhì)干細胞(Mesenchymal stem cells,MSCs)。MSCs一方面支持骨髓中造 血干細胞的生存、生長和分化。 另一方面, 經(jīng)許多體外實驗證明,它們是一種具有 多向分化潛能的細胞,具有極強的自我復制能力,在特定的條件下可以分化為中胚層 起源的多種組織和細胞(如:成骨細胞、軟骨細胞、肌細胞等),甚至可以分化為跨胚 層的細胞(如神經(jīng)細胞)。MSCs具有取材方便,不存在免疫排斥反應,而且易于進行 細胞擴增,易于臨床應用的特點,是一種重要的組織工程種子細胞。由于MSCs具有獨 特的細胞增殖分裂模式,使外源基因易于導入和表達,從而使其有望成為一種在人類 醫(yī)學上潛在的細胞治療和基因治療的靶細胞,在多種造血以外組織的缺陷性疾病、退 行性疾病和遺傳性疾病的細胞治療和基因治療中具有重要的應用前景。 但是MSCs在體外長期培養(yǎng)后也會遇到一些不利因素,諸如基因漂流,細胞衰老, 分化,表型不穩(wěn)定性,污染或者是培養(yǎng)箱故障等,而這些均會影響對MSCs的應用。因 此,適時地凍存MSCs是將來儲備細胞種子的一種重要手段。然而,凍存后MSCs的擴 增和分化能力如何,值得進一步研究。 本實驗對成人的骨髓間充質(zhì)干細胞(human mesenchyma].stem cells,hMSCs)進 行了體外分離、培養(yǎng)和初步的細胞誘導分化,并對其生長方式進行觀察,旨在建立一 種分離、培養(yǎng)hMSCs并誘導其向軟骨細胞、脂肪細胞和神經(jīng)細胞轉(zhuǎn)化的有效方法,為 hMSCs作為組織工程的細胞來源提供實驗依據(jù)。同時為檢測凍存的hMSCs在復蘇并傳 至15代后是否仍具有向軟骨、脂肪和神經(jīng)元多向分化的潛能,我們在通過黏附方法從 骨髓中獲得hMSCs并進行凍存的基礎(chǔ)上,復蘇細胞并傳至第15代,然后在相應的條件 培養(yǎng)液作用下依次誘導成軟骨細胞,脂肪細胞和神經(jīng)元細胞,觀察其形態(tài)學變化以及 在對應的誘導液的作用下膠原Ⅱ、三油酸甘油酯、巢蛋白(Nestin)和神經(jīng)元特異性烯 醇化酶(neuron-specific enolase,NSE)的表達,從而研究凍存細胞的擴增和分化潛 能。研究結(jié)果表明,凍存的hMSCs在復蘇后仍是具有單一特征的、均質(zhì)的細胞群。其
[Abstract]:The bone marrow contains not only hematopoietic stem cells which can differentiate into various blood cells, but also mesenchymal stem cells. MSCs can support the survival, growth and differentiation of hematopoietic stem cells. On the other hand, many experiments in vitro have proved that they are a kind of cells with multidirectional differentiation potential, and have a strong ability to replicate themselves. A variety of tissues and cells (such as osteoblasts, chondrocytes, myocytes, etc.) that can differentiate into mesoderm origin under certain conditions, and even cells that can be differentiated into transembryonic layer cells (e.g., neural cells. MSCs) have the advantages of being able to obtain materials, such as osteoblasts, chondrocytes, myocytes, and so on. It is an important seed cell of tissue engineering that there is no immune rejection, and it is easy to carry out cell expansion and is easy to be applied in clinic. Because MSCs have a unique cell proliferation and division mode, exogenous genes can be easily introduced and expressed, which makes MSCs a potential target cell for cell therapy and gene therapy in human medicine. MSCs have important application prospects in cell therapy and gene therapy for various diseases other than hematopoiesis, degenerative diseases and genetic diseases. However, MSCs may encounter some unfavorable factors after long-term culture in vitro. Gene drift, cell senescence, differentiation, phenotypic instability, contamination, or incubator failure all affect the use of MSCs. Therefore, cryopreservation of MSCs is an important method to store cell seeds in the future. However, the ability of expansion and differentiation of MSCs after cryopreservation is worthy of further study. In this experiment, MSCs from adult bone marrow mesenchymal stem cells (MSCs) were isolated in vitro, cultured and preliminarily differentiated. The purpose of this study was to establish an effective method to isolate, culture and induce the transformation of hMSCs into chondrocytes, adipocytes and nerve cells, and to provide experimental evidence for hMSCs as a cell source of tissue engineering. At the same time, in order to detect whether cryopreserved hMSCs still have the potential to differentiate into cartilage, fat and neurons after resuscitation and transmission to 15 generations, we obtained hMSCs from bone marrow by adhesion method and frozen them. The resuscitation cells were transferred to the 15th passage, then induced chondrocytes, adipocytes and neuronal cells in turn under the corresponding conditioned medium. The morphological changes of the cells and collagen 鈪，

本文編號：2002464