本文選題：間充質(zhì)干細(xì)胞　切入點(diǎn)：miRNA　出處：《第四軍醫(yī)大學(xué)》2008年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 背景microRNA(miRNA)是一類(lèi)非編碼蛋白并且參與轉(zhuǎn)錄后調(diào)節(jié)的單鏈小分子RNA(約20~25個(gè)堿基),對(duì)基因表達(dá)具有重要調(diào)控作用。近年來(lái)的研究表明在線蟲(chóng)、果蠅、小鼠、大鼠和人等多個(gè)物種中存在著一個(gè)龐大的miRNA家族,miRNA參與調(diào)控生物體的生長(zhǎng)發(fā)育、信號(hào)轉(zhuǎn)導(dǎo)、組織分化、疾病發(fā)生等,同時(shí)還決定了其它很多生物體發(fā)育和行為等的變化。骨髓間充質(zhì)干細(xì)胞在不同誘導(dǎo)條件下具有多向分化潛能,可分化成多種類(lèi)型的結(jié)締組織,如骨、軟骨、骨骼肌、肌腱、韌帶、真皮、脂肪和骨髓基質(zhì),也可分化成神經(jīng)系統(tǒng)的神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞等,而且來(lái)源廣泛,易于在體外擴(kuò)增,是目前組織工程中一種重要的種子細(xì)胞。 miRNA作為一個(gè)廣泛存在的可對(duì)基因表達(dá)進(jìn)行微調(diào)的分子,參與生物體的生長(zhǎng)、發(fā)育、衰老及死亡的調(diào)控,其在干細(xì)胞增殖及分化中的意義近年來(lái)開(kāi)始受到關(guān)注。miRNA在多種干細(xì)胞中均有表達(dá),并且通過(guò)負(fù)向調(diào)節(jié)干細(xì)胞中某些關(guān)鍵基因的表達(dá),對(duì)干細(xì)胞的自我更新和多向分化發(fā)揮作用。 目的通過(guò)檢測(cè)骨髓間充質(zhì)干細(xì)胞及其誘導(dǎo)分化的成骨細(xì)胞中miRNA基因的表達(dá)譜,篩選出誘導(dǎo)分化前后細(xì)胞表達(dá)的差異性miRNAs,尋找可能的人骨髓間充質(zhì)干細(xì)胞成骨分化特異性的miRNAs,為進(jìn)一步闡明miRNA在骨髓間充質(zhì)干細(xì)胞定向分化中的作用和意義奠定基礎(chǔ)。 方法(1)采用密度梯度離心法自骨髓中分離培養(yǎng)人骨髓間充質(zhì)干細(xì)胞,通過(guò)觀察原代及傳代MSCs的形態(tài)特點(diǎn),流式細(xì)胞儀檢測(cè)細(xì)胞表面標(biāo)志物,對(duì)人骨髓間充質(zhì)干細(xì)胞進(jìn)行鑒定。(2)采用地塞米松、β-甘油磷酸鈉和維生素C聯(lián)合誘導(dǎo)人MSCs向成骨細(xì)胞方向分化,通過(guò)ALP染色、茜素紅S法染色及電鏡觀察對(duì)誘導(dǎo)分化的成骨細(xì)胞進(jìn)行鑒定。(3)利用miRNA基因芯片技術(shù),檢測(cè)人骨髓間充質(zhì)干細(xì)胞及其誘導(dǎo)分化的成骨細(xì)胞中miRNAs的表達(dá)譜,并用SAM分析篩選出人MSCs和其誘導(dǎo)分化的成骨細(xì)胞表達(dá)的差異性miRNAs。(4)采用實(shí)時(shí)定量PCR和Northern blotting分析的方法,對(duì)miRNA基因芯片篩選出的人MSCs及其誘導(dǎo)分化的成骨細(xì)胞的部分差異miRNA基因進(jìn)行驗(yàn)證。 結(jié)果(1)經(jīng)密度梯度離心法分離貼壁培養(yǎng)所得到的細(xì)胞,通過(guò)流式細(xì)胞儀檢測(cè)的結(jié)果為:細(xì)胞表面標(biāo)志物CD29、CD44為陽(yáng)性,CD34、CD45為陰性,符合骨髓間充質(zhì)干細(xì)胞特征。(2)經(jīng)地塞米松、β-甘油磷酸鈉和維生素C聯(lián)合誘導(dǎo)14天后得到的細(xì)胞:ALP染色及茜素紅S法染色均陽(yáng)性;掃描電鏡顯示細(xì)胞多為多角形,伸突細(xì)長(zhǎng)且多,細(xì)胞相互交聯(lián),局部產(chǎn)生絮絲狀基質(zhì)成分;透射電鏡顯示細(xì)胞分裂相多見(jiàn),核仁明顯,大量的粗面內(nèi)質(zhì)網(wǎng)、高爾基體及線粒體,并可見(jiàn)部分溶酶體及分泌顆粒,細(xì)胞外基質(zhì)中出現(xiàn)膠原纖維及鈣鹽顆粒。符合成骨細(xì)胞的特征。(3)用miRNA基因芯片檢測(cè)了三組樣品中人骨髓間充質(zhì)干細(xì)胞及其誘導(dǎo)分化的成骨細(xì)胞miRNAs的表達(dá)譜,并篩選出了在人MSCs和其誘導(dǎo)分化的成骨細(xì)胞表達(dá)的差異性的miRNAs。其中在每組樣品中均篩選出了人MSCs和其誘導(dǎo)分化的成骨細(xì)胞的差異的miRNAs,如成骨細(xì)胞較MSCs高表達(dá)的miRNAs在三組樣品中分別為:36個(gè)、14個(gè)、8個(gè);成骨細(xì)胞較MSCs低表達(dá)的miRNAs在三組樣品中分別為:24個(gè)、27個(gè)、20個(gè)。但miRNAs存在很大的個(gè)體差異性,在三組樣品中有共同存在趨勢(shì)的MSCs和成骨細(xì)胞的差異miRNAs較少,如hsa-miR-30a-5p、hsa-miR-30c、hsa-miR-210、hsa-miR-31等。(4)針對(duì)hsa-miR-30a-5p和hsa-miR-210分別用Northern blotting和實(shí)時(shí)定量PCR的方法在第1組樣品中進(jìn)行驗(yàn)證,結(jié)果均與芯片檢測(cè)的結(jié)果一致。 結(jié)論(1)用密度梯度離心法可以從骨髓中分離得到人骨髓間充質(zhì)干細(xì)胞,并可在體外培養(yǎng)將其誘導(dǎo)分化為成骨細(xì)胞。(2)在人骨髓間充質(zhì)干細(xì)胞及其誘導(dǎo)分化的成骨細(xì)胞中均有大量miRNAs的表達(dá),但其表達(dá)在不同個(gè)體來(lái)源的細(xì)胞存在明顯的差異性。(3)在人骨髓間充質(zhì)干細(xì)胞和其誘導(dǎo)分化的成骨細(xì)胞中存在表達(dá)差異的miRNAs,它們可能對(duì)人骨髓間充質(zhì)干細(xì)胞的成骨誘導(dǎo)分化起著重要的調(diào)控作用,即所謂的人骨髓間充質(zhì)干細(xì)胞成骨分化特異性miRNA。
[Abstract]:Background microRNA (miRNA) is a small single stranded RNA for a class of non protein encoding and participate in the post transcriptional regulation of the (about 20~25 nucleotides), plays an important role in regulating gene expression. Recent studies showed that C. elegans, Drosophila, mouse, rat and human and other species in the existence of a huge miRNA the miRNA family, is involved in the regulation of growth and development of organism, signal transduction, tissue differentiation, occurrence of disease, but also decide the change of many other organism development and behavior. Bone marrow mesenchymal stem cells with multi-directional differentiation potential under different induction conditions, can differentiate into various types of connective tissue, such as bone, cartilage, skeletal muscle, tendon, ligament, leather, fat and bone marrow, can differentiate into neurons and glial cells, and a wide range of sources, easy in vitro amplification, is an important seed in tissue engineering at present Cells.
MiRNA as a widespread of gene expression in fine-tuning the molecule, involved in organism growth, development, aging and death in the regulation of stem cell proliferation and differentiation in significance in recent years, researchers have begun to focus on.MiRNA in a variety of stem cells expressed, and through the negative regulation of stem cell gene expression of some key the role of stem cell self-renewal and differentiation.
The spectrum of miRNA expression in osteoblasts in gene detection of bone marrow mesenchymal stem cells and their differentiation, screened before and after differentiation of miRNAs cells the expression of differences, to find possible human bone marrow mesenchymal stem cells differentiation of bone specific miRNAs, to further clarify the role of bone marrow and miRNA the significance of cell differentiation in mesenchymal stem lay the foundation.
Methods (1) isolated from bone marrow cultured human bone marrow mesenchymal stem cells by density gradient centrifugation, the morphological characteristics of primary and passage MSCs, flow cytometry was used to detect cell surface markers and identification of human bone marrow mesenchymal stem cells. (2) using dexamethasone, beta sodium glycerophosphate and vitamin C and induce MSCs to differentiate into osteoblasts by ALP staining, alizarin red S staining and electron microscopy were used to identify the osteoblast differentiation. (3) using miRNA gene chip technology, detection of human bone marrow mesenchymal stem cells and differentiation into bone miRNAs expression cells in the spectrum, and SAM analysis showed that MSCs and the differentiation of osteoblasts in different expression of miRNAs. (4) by using the method of real-time quantitative analysis of PCR and Northern blotting, screened by miRNA gene chip MSCs and its differentiation into The partial difference of bone cells was verified by miRNA gene.
Results (1) the adherent cells obtained by density gradient centrifugation, by flow cytometry results: cell surface markers CD29, CD34, CD44 positive, CD45 negative with bone marrow mesenchymal stem cells. (2) by dexamethasone, beta glycerol phosphate sodium and vitamin C combined with 14 days after the induction of the cells: ALP staining and alizarin red S staining were positive; scanning electron microscopy showed that the cells were polygonal, slender and stretching process, cells interconnected, local flocculation of filamentous matrix components; transmission electron microscopy revealed that the cell division phase, obvious nucleolus, a lot of rough the endoplasmic reticulum, Golgi apparatus and mitochondria and secretory granules, and the visible portion of lysosomes, collagen fibrils and calcium salt particles in the extracellular matrix. In line with the characteristics of osteoblasts. (3) in the three groups of samples of human bone marrow mesenchymal stem cells and its detection by miRNA gene chip Spectrum miRNAs expression of osteoblast differentiation, and screened in MSCs and the differentiation of osteoblasts difference expression of miRNAs. in each sample were screened out MSCs and its differentiation into different bone cells of miRNAs, such as osteoblasts with high expression of MSCs the miRNAs samples in three groups respectively: 36, 14, 8; low expression of bone cells than MSCs miRNAs in the three groups of samples were 24, 27, 20. But the miRNAs are difference, there are differences in common trend MSCs and osteogenesis the miRNAs cell is less, the samples in three groups such as hsa-miR-30a-5p, hsa-miR-30c, hsa-miR-210, hsa-miR-31 et al. (4) according to the method of hsa-miR-30a-5p and hsa-miR-210 respectively by Northern blotting and real-time quantitative PCR in first samples to verify the results with the chip detection results.
Conclusion (1) human bone marrow mesenchymal stem cells isolated from bone marrow by density gradient centrifugation, and were cultured in vitro and differentiate into bone cells. (2) were expressed in bone cells in a large number of miRNAs into human bone marrow mesenchymal stem cells and their differentiation, but the there are obvious differences in the expression of different individual cells. (3) in human bone marrow mesenchymal stem cells and their differentiation into the differential expression of miRNAs in bone cells, they may be important for regulation of human bone marrow mesenchymal stem cells osteoblast differentiation plays, the so-called human bone marrow mesenchymal stem cells into bone cells specific miRNA.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R329

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