發(fā)布時(shí)間：2018-09-19 07:24

【摘要】： 神經(jīng)干細(xì)胞(Neural stem cells,NSCs)作為一種具備自我更新能力及多向分化潛能的細(xì)胞,自被發(fā)現(xiàn)以來,就因其對神經(jīng)系統(tǒng)的修復(fù)能力而受到廣大學(xué)者的關(guān)注。但是神經(jīng)干細(xì)胞的來源受限及數(shù)量不足是臨床應(yīng)用的最大障礙。采用體外培養(yǎng)以實(shí)現(xiàn)大規(guī)模擴(kuò)增是這個(gè)問題的一種有效解決辦法。然而,在二維神經(jīng)球懸浮培養(yǎng)條件下,由于球體自身結(jié)構(gòu)的制約,隨著神經(jīng)球的直徑增長,營養(yǎng)物質(zhì)由外及內(nèi)傳遞以及細(xì)胞代謝物由內(nèi)及外擴(kuò)散均受到影響。考慮到生物支架材料-膠原(Collagen I,COL I)在干細(xì)胞體外大規(guī)模培養(yǎng)領(lǐng)域的應(yīng)用,如果把細(xì)胞接種在由膠原制成的三維支架材料中進(jìn)行培養(yǎng),將會(huì)成為一個(gè)更有效的神經(jīng)干細(xì)胞培養(yǎng)方式。 為了深入了解神經(jīng)干細(xì)胞在由膠原制成的三維支架材料中的生長狀況,本文將實(shí)驗(yàn)數(shù)據(jù)與傳質(zhì)代謝方程相結(jié)合,通過求解數(shù)學(xué)模型,計(jì)算支架內(nèi)各種代謝物質(zhì)的濃度分布,進(jìn)而篩選出最有利于培養(yǎng)神經(jīng)干細(xì)胞的膠原支架厚度和適宜的細(xì)胞接種密度。 首先,從E14小鼠胚胎海馬腦組織中獲得原代細(xì)胞,體外無血清懸浮培養(yǎng),利用誘導(dǎo)分化和免疫熒光對傳代細(xì)胞進(jìn)行生物學(xué)鑒定,同時(shí)測定單個(gè)神經(jīng)干細(xì)胞對溶解氧的代謝參數(shù)。其次,檢測細(xì)胞-膠原支架的葡萄糖、乳酸代謝情況,與傳質(zhì)代謝模型計(jì)算結(jié)果進(jìn)行比對,驗(yàn)證模型的可靠性。最后,通過計(jì)算不同厚度膠原支架內(nèi)代謝物質(zhì)的濃度分布,選擇最佳成膠工藝參數(shù)。 實(shí)驗(yàn)結(jié)果表明:①所培養(yǎng)的細(xì)胞具有增殖能力和分化潛能,符合神經(jīng)干細(xì)胞的定義,可用于單個(gè)神經(jīng)干細(xì)胞氧代謝的研究。②對所培養(yǎng)的神經(jīng)干細(xì)胞氧代謝的測定結(jié)果顯示,單個(gè)神經(jīng)干細(xì)胞的氧代謝速率為2.9×10~(-14)mmol/cell/s。③膠原的終濃度不宜低于1mg/ml,否則無法滿足三維支架制備過程中對機(jī)械強(qiáng)度的要求。④在葡萄糖初始濃度5.08×10~(-2)mmol/ml、培養(yǎng)基液層厚度5mm、細(xì)胞接種密度1×10~6cells/ml為培養(yǎng)條件下,2mm厚度的膠原支架完全能夠滿足細(xì)胞正常生長對葡萄糖、乳酸和溶解氧濃度的要求。 結(jié)合模型計(jì)算,最終確定神經(jīng)干細(xì)胞接種于膠原支架內(nèi)進(jìn)行體外三維培養(yǎng)的適宜條件為:凝膠層厚度2mm、培養(yǎng)基液層厚度5mm、細(xì)胞接種密度2×10~5cells/ml。
[Abstract]:Neural stem cell (Neural stem cells,NSCs), as a kind of cells with self-renewal ability and multi-differentiation potential, has attracted much attention since it was discovered because of its ability to repair the nervous system. However, limited sources and insufficient number of neural stem cells are the biggest obstacle to clinical application. In vitro culture for large-scale amplification is an effective solution to this problem. However, under the condition of 2-D suspension culture, due to the restriction of the structure of the sphere itself, with the increase of the diameter of the ball, the transfer of nutrients from the outside and the diffusion of the metabolites from the inside and outside are affected. Considering the application of biological scaffold collagen (Collagen I) in the field of stem cell mass culture in vitro, if the cells were cultured in a three-dimensional scaffold made of collagen, It will be a more effective way to culture neural stem cells. In order to understand the growth of neural stem cells (NSCs) in three dimensional scaffolds made of collagen, this paper combines the experimental data with the mass transfer equation, and calculates the concentration distribution of various metabolites in the scaffold by solving the mathematical model. The thickness of collagen scaffold and the suitable seeding density were selected. First, primary cells were obtained from the hippocampus of E14 mouse embryos, and cultured without serum in vitro. The passage cells were identified by induction differentiation and immunofluorescence, and the metabolic parameters of dissolved oxygen by single neural stem cells were measured. Secondly, the glucose and lactate metabolism of the cell-collagen scaffold was detected and compared with the results of mass transfer model to verify the reliability of the model. Finally, by calculating the concentration distribution of metabolites in collagen scaffolds with different thickness, the optimum gelling process parameters were selected. The results show that the cells cultured at 1: 1 have the ability of proliferation and differentiation, which accord with the definition of neural stem cells, and can be used in the study of oxygen metabolism of single neural stem cells. The oxygen metabolism rate of single neural stem cells is 2.9 脳 10 ~ (-14) mmol/cell/s.3 collagen concentration should not be less than 1 mg / ml, otherwise, it can not meet the requirement of mechanical strength in the process of three-dimensional scaffold preparation. 4 at the initial glucose concentration of 5.08 脳 10 ~ (-2) mmol/ml, medium, the thickness of the liquid layer is 5 mm and the thickness is fine. The collagen scaffold with a thickness of 2 mm and a cell density of 1 脳 10~6cells/ml could completely satisfy the normal growth of the cells to glucose. Lactic acid and dissolved oxygen concentration requirements. Combined with the model calculation, the suitable conditions for the three-dimensional culture of neural stem cells in collagen scaffold were determined as follows: the thickness of gel layer was 2 mm, the thickness of medium was 5 mm, and the cell density was 2 脳 10 ~ (5) cells / ml.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 呂艷玲;胚胎大鼠海馬神經(jīng)干細(xì)胞的擴(kuò)增及其與三維微小凹圖式復(fù)合的研究[D];重慶大學(xué);2010年

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本文編號：2249436