兔传代骨髓间充质干细胞的筛选及其成骨能力的研究

发布时间：2017-12-27 23:01

本文关键词：兔传代骨髓间充质干细胞的筛选及其成骨能力的研究　出处：《桂林医学院》2013年硕士论文　论文类型：学位论文

【摘要】：目的：1.本研究从兔骨髓液中分离骨髓间充质干细胞（BMSCs），进行体外培养，旨在建立一套行之有效的体外分离，培养BMSCs的方法。2.通过各代观察BMSCs的形态，检测不同代BMSCs的细胞增殖周期和细胞生长周期，从而了解各代BMSCs的生物学性状以及初步确定作为理想的组织工程种子细胞的理想代数。3.BMSCs在成骨诱导液的诱导下向成骨细胞特定分化，通过检测成骨细胞特有指标Ⅰ型胶原，，碱性磷酸酶和钙结节的表达，了解BMSCs向成骨细胞方向分化的能力。方法：1.利用骨穿针在兔股骨抽取骨髓液，利用密度梯度离心法联合贴壁细胞筛选法对BMSCs进行体外培养，观察细胞的生长情况。2.观察各代细胞形态并且测定各代细胞的增殖周期和细胞生长周期，了解各代细胞的生物学性状。3.在体外利用成骨诱导液（完全培养基中加入地塞米松浓度为10-8mol/L、β-磷酸甘油钠10mmol/L、维生素C50ug/ml），在诱导的第7天，第14天和第21天分别检测Ⅰ型胶原，碱性磷酸酶（ALP）,钙结节表达。研究BMSCs向成骨细胞分化的能力。结果：1.利用密度梯度离心法联合贴壁筛选法分离出了大量具有活性的BMSCs。接种48小时后观察，见少量细胞呈长梭性，大部分为短梭形和多边形。接种72小时观察，贴壁细胞明显增多，呈长梭形。接种第5天，细胞增殖明显，并且可见若干细胞团落形成。接种第7天，达到融合。2.第1，5，10代细胞经MTT和流式细胞仪检测，第1代和第5代的增殖能力和生长周期无显著性差异（P0.05），第10代细胞与第1代细胞，第5代细胞之间的增殖能力和细胞生长周期较前两代有明显的差别（P0.05）。3．BMSCs向成骨方向分化：贴壁细胞随着时间的延长，由最初的长梭状，逐渐向多角形发展。诱导组细胞呈现接触性抑制，正常组呈现重叠性生长。Ⅰ型胶原染色显示诱导组细胞胞质呈现棕黄色，正常组胞质未染色。ALP酶标法显示诱导组较正常组的含量明显增加（P0.05）。诱导组钙结节染色呈红色，正常组未染色。显示BMSCs在特定条件下可以很好的向成骨细胞分化。结论：1.通过利用密度梯度离心法联合贴别筛选法可以获得较高纯度和活性的BMSCs。2.通过对各代BMSCs形态学的观查，细胞增殖周期和细胞生长周期的检测，证实第1，5代细胞之间的生物学特性无明显的区别，第10代细胞增值减慢，处于非分裂期的细胞明显增多，与第1，5代细胞之间有明显的差别，且第5细胞已成为单一细胞系，所以确立第5代细胞可以成为组织工程中提供良好的种子细胞。3.通过利用成骨诱导液对BMSCs向成骨细胞诱导，并对成骨细胞特定指标Ⅰ型胶原，ALP,钙结节检测的结果显示，第5代BMSCs在含有成骨诱导液条件下可以向成骨细胞分化，并且具有很强的分化能力。
[Abstract]:Objective: 1.. In this study, bone marrow mesenchymal stem cells (BMSCs) were isolated from rabbit bone marrow and cultured in vitro. The aim was to establish an effective method for isolating and culturing BMSCs in vitro. 2., we can observe the morphology of BMSCs through various generations, detect cell proliferation cycle and cell growth cycle of different generations of BMSCs, so as to understand the biological characteristics of each generation of BMSCs and preliminarily determine the ideal algebra as an ideal tissue engineering seed cell. 3.BMSCs differentiated into osteoblasts under the induction of osteogenic induction. By detecting the expression of type I collagen, alkaline phosphatase and calcium nodules in osteoblasts, we could understand the ability of BMSCs to differentiate into osteoblasts. Methods: 1. using bone needle in rabbit femoral bone marrow, using density gradient centrifugation and adherent cells were cultured in vitro screening method of BMSCs, observe the cell growth. 2. observe the cell morphology of each generation and determine the cycle of cell proliferation and cell growth cycle, and understand the biological characters of each generation. 3. in vitro using osteogenic medium (complete medium with dexamethasone concentration 10-8mol/L, beta glycerophosphate sodium 10mmol/L, vitamin C50ug/ml), on the seventh day of induction, fourteenth days and twenty-first days respectively, type I collagen, alkaline phosphatase (ALP), the expression of calcium nodules. To study the ability of BMSCs to differentiate into osteoblasts. Results: 1. a large number of active BMSCs were separated by the combination of density gradient centrifugation and wall screening. After 48 hours of inoculation, a small number of cells were found to be spindle, most of which were short shuttle and polygon. After 72 hours of inoculation, the adherent cells were significantly increased, showing a long spindle shape. After fifth days of inoculation, the cell proliferation was obvious, and a number of cell clusters were formed. Inoculation for seventh days to achieve fusion. 2., first, 5, 10 generation cells by MTT and flow cytometry, no significant difference between the first and fifth generation of proliferation and growth cycle (P0.05), the tenth generation of cells and cells of the first generation, fifth generation of cell proliferation and cell cycle compared with the previous two generations have obvious difference (P0.05). 3.BMSCs differentiated into osteogenic direction: the adherent cells developed from the initial long spindle shape to polygon with the prolongation of time. The cells in the induced group showed contact inhibition, and the normal group showed overlapping growth. Type I collagen staining showed that the cytoplasm of the induced group showed brown yellow, and the normal cytoplasm was not stained. The ALP enzyme labeling method showed that the content of the induced group was significantly higher than that in the normal group (P0.05). The coloring of calcium nodules in the induced group was red, and the normal group was not stained. It is shown that BMSCs can be well differentiated into osteoblasts under specific conditions. Conclusion: 1. the high purity and activity of BMSCs can be obtained by the combination of density gradient centrifugation and the combination screening method. Based on the 2. generation BMSCs morphological observation, detection, cell cycle and cell growth cycle was confirmed in first, no significant difference between the biological characteristics of cells of the 5 generation, tenth generation cell proliferation slowed in nondividing cells increased significantly, and the first, there were significant differences between the 5 generation cells, and fifth cells have become single cells, so the establishment of the fifth generation cells can be provided good seed cells in tissue engineering. 3. through the use of osteogenic medium on BMSCs osteogenic induction, and osteoblast specific index of collagen, ALP, calcium nodules detection results showed that the fifth generation BMSCs can differentiate into osteoblast in osteogenic medium containing conditions, and has a strong ability of differentiation.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329.2

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