兔骨髓间充质干细胞的体外分离培养和成骨诱导

发布时间：2018-01-21 02:15

本文关键词： 骨髓间充质干细胞细胞培养成骨诱导　出处：《山东大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的:本实验从骨髓中分离间充质干细胞(MSCs)体外扩增培养,观察MSCs的形态,检测不同因素对MSCs生长的影响,测定生长曲线。并体外诱导MSCs向成骨细胞方向分化,检测成骨特有的骨钙素及Ⅰ型胶原的表达,从而了解MSCs的生物学形状,探索MSCs的生长条件。方法:抽取兔髂骨骨髓,利用Percoll分离液(1.073g/ml)进行密度梯度离心法从骨髓中分离骨髓间充质干细胞,体外培养扩增,观察其形态学变化,测定MSC的生长动力学及对MSC生长的影响因素的分析,了解MSCs的生物学性状,研究MSCs的体外生长规律。同时体外分离诱导培养MSCs向成骨细胞分化(培养液中加入地塞米松终浓度为10~(-8)mol/L-、β—甘油磷酸钠为10mmol/L、维生素C为50μg/mL),通过检测碱性磷酸酶表达,Ⅰ型胶原分泌,骨钙素生成对其体外诱导成骨能力进行定性检测和观察,研究细胞生物学特性及其体外成骨能力。结果:1、分离获取了较高纯度的MSCs,保持了细胞的活性。Percoll分离液(1.073g/ml)分离的骨髓单个核细胞24小时后贴壁,细胞呈圆形,48—72小时后呈纺锤形,梭形,增值迅速,原代培养月10—14天左右可以达到80—90%融合,传代细胞24小时完全贴壁,5—7天达到80—90%融合,可传代至10代以上,经传代后细胞增值能力有所下降,MSCs细胞逐渐出现衰老现象。细胞老化的表现为:细胞生长速度缓慢。细胞胞浆中出现多量空泡及颗粒样物质,细胞碎片的增多。在该培养条件下,原代
[Abstract]:Objective: to investigate the effects of different factors on the growth of mesenchymal stem cells (MSCs) in vitro, to observe the morphology of MSCs and to investigate the effect of different factors on the growth of MSCs. The growth curve was measured and MSCs was induced to differentiate into osteoblasts in vitro. The expression of osteocalcin and type I collagen was detected in order to understand the biological shape of MSCs. To explore the growth conditions of MSCs. Methods: rabbit iliac bone marrow was extracted. Bone marrow mesenchymal stem cells were isolated from bone marrow by density gradient centrifugation with Percoll (1.073 g / ml) and cultured in vitro. The morphological changes were observed, the growth kinetics of MSC and the influencing factors on the growth of MSC were analyzed, the biological characters of MSCs were understood, and the rule of MSCs growth in vitro was studied. At the same time, the differentiation of MSCs into osteoblasts was induced in vitro (the final concentration of dexamethasone was 10 ~ 8mol / L ~ (-)). 尾 -glycerophosphate was 10 mmol / L, vitamin C was 50 渭 g / mL. Type I collagen was secreted by detecting the expression of alkaline phosphatase. Osteocalcin production was used to detect and observe the osteogenic ability of osteoblasts in vitro and to study the biological characteristics and osteogenic ability of osteoblasts in vitro. Results: 1, high purity MSCs was obtained. The bone marrow mononuclear cells were adherent to the wall after 24 hours, and the cells were fusiform and fusiform after 48-72 hours. The primary culture could reach 80-90% fusion in 10-14 days, and 80-90% fusion could be achieved on 5-7 days after 24 hours of passage, and it could be subcultured to more than 10 generations. After passage, the proliferation ability of MSCs cells decreased gradually. The aging of MSCs cells showed that the growth rate of cells was slow, and a lot of vacuoles and granular-like substances appeared in the cytoplasm of MSCs cells. The increase of cell fragments. In this culture condition, the primary culture
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329.2

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