密度梯度离心及贴壁分离筛选相结合分离培养大鼠骨髓间充质干细胞

发布时间：2018-02-25 16:28

本文关键词： 骨髓间质干细胞细胞培养的大鼠 Sprague-Dawley 干细胞骨髓干细胞贴壁分离筛选法骨髓间充质干细胞生物学特性 SD大鼠细胞形态细胞周期增殖　出处：《中国组织工程研究》2014年28期 　论文类型：期刊论文

【摘要】：背景:在正常情况下骨髓来源间充质干细胞含量很少,且易与其他细胞相混杂,因此,建立一种简便可行的体外培养扩增方法,获得大量稳定的骨髓间充质干细胞具有重要的理论意义和应用价值。目的:建立一种简便可行的体外培养扩增方法,获得大量稳定的骨髓间充质干细胞。方法:采用密度梯度离心法及贴壁分离筛选法相结合体外分离培养SD大鼠骨髓间充质干细胞。倒置光学显微镜下观察细胞形态变化,透射电镜观察细胞亚微结构,锥虫蓝拒染法计算活细胞数,绘制细胞生长曲线,流式细胞仪分析细胞周期,免疫细胞化学检测c-kit和CD45的表达,流式细胞仪分析CD45的表达情况。结果与结论:①接种后24 h倒置光学显微镜下可见有细胞贴壁并伸出伪足,4 d时可见有细胞集落形成,14 d时细胞可达到90%融合。经传代后细胞趋于一致,为纤维样细胞,呈漩涡或火焰状生长。②透射电镜下可见第3代骨髓间充质干细胞体积较小核大,核仁明显。染色质分布稀疏,电子密度低。细胞表面有微绒毛,胞质稀松,内有丰富核糖体,而内质网、线粒体、高尔基复合体等细胞器少见,提示细胞处于原始未分化状态。③第3代骨髓间充质干细胞接种后第1天细胞数量有所减少,第2天细胞开始增长,第3天细胞进入指数增生期,第7天进入平台期,第9天细胞数量开始下降,绘制的生长曲线呈"S"形。④第3代骨髓间充质干细胞经DNA染色后采用流式细胞仪测定其DNA含量证明S期细胞比率为21.1%。⑤免疫细胞化学染色和流式细胞术证明c-kit阳性细胞比率为53.3%,CD45表达阳性率为1.68%。⑥骨髓间充质干细胞向成骨细胞诱导分化16 d后,细胞的胞体呈椭圆形,有短突起彼此相连,胞浆较暗,提示可能富含粗面内质网和高尔基复合体,并具有分泌类骨质的功能。结果证明该方法获得的大鼠骨髓间充质干细胞生长稳定,增殖活跃。
[Abstract]:Background: under normal conditions, mesenchymal stem cells derived from bone marrow have little content and are easily mixed with other cells. Therefore, a simple and feasible method for culture and amplification in vitro is established. Obtaining a large number of stable bone marrow mesenchymal stem cells is of great theoretical significance and practical value. Objective: to establish a simple and feasible method for in vitro culture and amplification of bone marrow mesenchymal stem cells. A large number of stable bone marrow mesenchymal stem cells were obtained. Methods: bone marrow mesenchymal stem cells of SD rats were isolated and cultured in vitro by density gradient centrifugation and adherent separation screening. The ultrastructure of cells was observed by transmission electron microscope, the number of living cells was calculated by trypanosome blue exclusion method, the cell growth curve was drawn, the cell cycle was analyzed by flow cytometry, and the expression of c-kit and CD45 was detected by immunocytochemistry. Results and conclusion the adherent cells were observed under inverted optical microscope 24 h after inoculation and the cells could reach 90% fusion at 14 d after 14 d of colony formation. After passage, the cells tend to converge. The mesenchymal stem cells of the third generation were larger in size, obvious in nucleoli, sparse in chromatin distribution, low in electron density, and microvilli on the surface of the cells. There were abundant ribosomes in the cells, but few organelles such as endoplasmic reticulum, mitochondria and Golgi complex, which indicated that the number of cells in the third generation of bone marrow mesenchymal stem cells was decreased on the first day after inoculation. On the second day, the cells began to increase, on the third day the cells entered the exponential proliferative phase, on the 7th day into the plateau phase, and on the 9th day, the number of cells began to decrease. The growth curve of the third generation bone marrow mesenchymal stem cells was "S". 4. After DNA staining, flow cytometry was used to determine its DNA content. The ratio of S phase cells was 21.1.5 immunocytochemical staining and flow cytometry was used to prove c-kit positive fine cells. The positive rate of CD45 expression was 1.68.6 bone marrow mesenchymal stem cells differentiated into osteoblasts 16 days after induction. The cell body is elliptical, with short processes connected to each other, and the cytoplasm is dark, suggesting that the cells may be rich in rough endoplasmic reticulum and Golgi complex. The results showed that the bone marrow mesenchymal stem cells obtained by this method were stable in growth and active in proliferation.
【作者单位】：武警后勤学院临床医学系实验技术教研室;
【分类号】：R329

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