四点弯曲应力刺激对人骨髓间充质干细胞增殖活性的影响
发布时间：2018-09-11 08:32
【摘要】：目的:观察四点弯曲应力刺激对人骨髓间充质干细胞的增殖活性以及细胞周期的影响。 方法:实验于2010.9—2011.3在大连医科大学附属第一医院中英再生医学中心完成。 1.使用全骨髓培养法分离人骨髓间充质干细胞。进行原代培养,扩增后备用。 2.利用四点弯曲应力细胞加载仪(四川大学华西医学院与电子科技大学联合研制,专利号:(CN2534576 and CN1425905),取第4~ 6代细胞接种于加力板(BD No.353135),行爬片处理。分组:空白组,不加力;实验组,实验组频率均为0.5HZ,加力60min,分别受张应力1000μstrain、3000μstrain、5000μstrain的机械信号刺激,空白组不受力学信号刺激,其他培养条件与实验组一致。 3.细胞行力学干预后,用倒置相差显微镜观察细胞形态、排列。分别用四甲基偶氮唑细胞计数法、流式细胞仪周期分析、共聚焦显微镜检测钙离子含量来测量细胞增殖和细胞周期。所有实验均重复三次。 4.统计学处理:实验数据均以均数±标准差来表示,使用SPSS16.0软件包进行单因素方差分析,P0.05具有统计学意义,P0.01具有显著统计学意义。 结果: 1.利用离心法结合全骨髓培养法培养细胞增殖迅速,传代稳定。 2.四点弯曲应力细胞加载仪性能稳定,准确,培养板未出行裂痕,变形等情况。细胞接种加力板后12 ~24h贴壁,贴壁良好,24 h后细胞覆盖率约达80%~90%,无细菌污染现象,可适用于应力刺激体外细胞效应的实验研究。 3.通过四甲基偶氮唑法细胞计数检测细胞增殖、流式细胞技术检测细胞周期及PI指数,激光共聚焦显微镜钙离子探针检测细胞内钙离子浓度,发现适当大小的机械应力刺激对人骨髓间充质干细胞增殖具促进作用,其中3000μstrain的应力刺激作用最明显,过强的机械应力刺激(5000μstrain)对细胞不具有促增殖效应,并可能存在抑制作用。 结论:适当的机械应力刺激可以促进人骨髓间充质干细胞增殖。
[Abstract]:Aim: to observe the effects of four point bending stress stimulation on proliferation activity and cell cycle of human bone marrow mesenchymal stem cells. Methods: the experiment was carried out in the first affiliated Hospital of Dalian Medical University. Human bone marrow mesenchymal stem cells were isolated by whole bone marrow culture. The primary culture was carried out, and then the amplification was carried out. 2. 2. A four-point bending stress cell loading instrument (Huaxi Medical College of Sichuan University and University of Electronic Science and Technology, patent number: (CN2534576 and CN1425905) was used to inoculate the 4th to 6th generation cells on the BD No.353135. The frequency of the experimental group was 0.5 HZ, and the experimental group was exposed to mechanical signal of 1000 渭 strain,3000 渭 strain,5000 渭 strain for 60 minutes, while the blank group was not stimulated by mechanical signal, and the other culture conditions were the same as that of the experimental group. After mechanical intervention, the morphology and arrangement of cells were observed by inverted phase contrast microscope. The cell proliferation and cell cycle were measured by the method of tetramethylazo cell count, flow cytometry and confocal microscope. All experiments were repeated three times. 4. Statistical processing: the experimental data were expressed as mean 卤standard deviation, using SPSS16.0 software package for univariate ANOVA (P0.05) had statistical significance (P0.01). Results: 1. The cell proliferation was rapid and the passage was stable by centrifugation and whole bone marrow culture. 2. The four-point bending stress cell loader is stable, accurate, no crack, deformation and so on. The cell coverage rate was about 80% after 24 hours of good adhesion, and there was no bacterial contamination, which could be applied to the experimental study of stress stimulated cell effect in vitro. Cell cycle and PI index were measured by flow cytometry and intracellular calcium concentration was detected by laser confocal microscope calcium probe. It was found that mechanical stress stimulation of appropriate size could promote the proliferation of human bone marrow mesenchymal stem cells, of which 3000 渭 strain stress stimulation was the most obvious, and excessive mechanical stress stimulation (5000 渭 strain) had no proliferative effect on the cells, and might have inhibitory effect on the proliferation of human bone marrow mesenchymal stem cells. Conclusion: proper mechanical stress stimulation can promote the proliferation of human bone marrow mesenchymal stem cells.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2236163
[Abstract]:Aim: to observe the effects of four point bending stress stimulation on proliferation activity and cell cycle of human bone marrow mesenchymal stem cells. Methods: the experiment was carried out in the first affiliated Hospital of Dalian Medical University. Human bone marrow mesenchymal stem cells were isolated by whole bone marrow culture. The primary culture was carried out, and then the amplification was carried out. 2. 2. A four-point bending stress cell loading instrument (Huaxi Medical College of Sichuan University and University of Electronic Science and Technology, patent number: (CN2534576 and CN1425905) was used to inoculate the 4th to 6th generation cells on the BD No.353135. The frequency of the experimental group was 0.5 HZ, and the experimental group was exposed to mechanical signal of 1000 渭 strain,3000 渭 strain,5000 渭 strain for 60 minutes, while the blank group was not stimulated by mechanical signal, and the other culture conditions were the same as that of the experimental group. After mechanical intervention, the morphology and arrangement of cells were observed by inverted phase contrast microscope. The cell proliferation and cell cycle were measured by the method of tetramethylazo cell count, flow cytometry and confocal microscope. All experiments were repeated three times. 4. Statistical processing: the experimental data were expressed as mean 卤standard deviation, using SPSS16.0 software package for univariate ANOVA (P0.05) had statistical significance (P0.01). Results: 1. The cell proliferation was rapid and the passage was stable by centrifugation and whole bone marrow culture. 2. The four-point bending stress cell loader is stable, accurate, no crack, deformation and so on. The cell coverage rate was about 80% after 24 hours of good adhesion, and there was no bacterial contamination, which could be applied to the experimental study of stress stimulated cell effect in vitro. Cell cycle and PI index were measured by flow cytometry and intracellular calcium concentration was detected by laser confocal microscope calcium probe. It was found that mechanical stress stimulation of appropriate size could promote the proliferation of human bone marrow mesenchymal stem cells, of which 3000 渭 strain stress stimulation was the most obvious, and excessive mechanical stress stimulation (5000 渭 strain) had no proliferative effect on the cells, and might have inhibitory effect on the proliferation of human bone marrow mesenchymal stem cells. Conclusion: proper mechanical stress stimulation can promote the proliferation of human bone marrow mesenchymal stem cells.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
【参考文献】
相关期刊论文 前4条
1 刘长剑;汤欣;;Pannexins——新发现的机械刺激敏感通道蛋白[J];长春中医药大学学报;2010年02期
2 戚孟春,胡静,邹淑娟,韩力赤,罗恩;大鼠骨髓间充质干细胞和颅骨成骨细胞在张应力下细胞骨架的改变[J];华西口腔医学杂志;2005年02期
3 赵光举;卢中秋;姚咏明;;哺乳动物细胞线粒体融合-分裂与钙离子信号的关系[J];生理科学进展;2010年03期
4 孙晓江;戴\戎;汤亭亭;;流体剪切应力对骨细胞分子活动的影响[J];医用生物力学;2007年01期
，本文编号：2236163
本文链接：https://www.wllwen.com/xiyixuelunwen/2236163.html
最近更新
教材专著
