周期性牵拉应力下三维培养的成骨样细胞的生物力学效应及其机制的研究
发布时间：2019-06-18 19:07
【摘要】：1 成骨样细胞的三维培养及牵拉应力下形态学改变 目的:研究克隆的鼠颅骨成骨样细胞(colonal murine osteoblast-like cell line MC3T3-E1)三维培养方法及在特定的周期性牵拉应力刺激下形态学改变。骨组织工程的关键在于创建类似活体生理环境的细胞体外培养体系。以往的体外细胞培养为单层、即二维细胞培养,这与细胞在活体的生存环境有很大差异。目前,建立三维细胞培养体系是骨组织工程的研究热点,不同的组织细胞对三维结构载体的要求条件存在差异。本实验选择克隆的鼠颅骨成骨样细胞作为种子细胞,创建三维结构培养体系,研究细胞培养过程中的细胞演变,并进一步在动力条件下观察细胞的形态学改变,为进一步探讨成骨细胞受到机械牵拉应力刺激后的分子生物学效应及信号传导机制奠定基础,从而指导临床中骨折的治疗。 方法: 以明胶海绵(2cmx2cmx0.25cm)作为MC3T3-E1 的三维培养支架,明胶海绵来源于纯化的猪皮肤胶原蛋白(Gelfoam; Upjohn, Kalamazoo, MI), 每块明胶海绵种植细胞悬液100μl,细胞数目为1.25×10~5个。生物牵拉装置(Bio-Stretch System; ICCT Technologies, Inc., Canada)包括一个程序控制的脉冲计时器、控制器、双向调节直流电源和一套带有平台的螺线电导管。放有明胶海绵的培养皿置于每一个电导管的前方。明胶海绵的一端固定于皿底,另一端用塑料夹包裹一个金属棒。供电后通过线圈的电流产生磁场,吸引金属棒朝线圈方向移动,拉伸明胶海绵。断电后,明胶海绵回缩为原来的长度。胶原海绵拉伸度为5%、作用频率为60cycles/min、作用时间为15min/h。牵拉后2d、4d、6d、8d、10d 分别从牵拉组和对照组中各取3 个样本,行细胞计数。将对照组和牵拉组各时相的每快明胶海绵均匀切成四份,布安氏液(Bouin’s fluid)固定48h,然后按以下程序制作切片:脱水、石蜡包埋:石蜡切片为6μm;脱蜡、苏木精伊红染色。HE 染色光镜下观察其形态学改变。所得数据采用SAS6.12统计软件进行两样本t 检验。
[Abstract]:1 three-dimensional culture of osteoblast-like cells and morphological changes under traction stress objective: to study the three-dimensional culture method of cloned rat skull osteoblast-like cells (colonal murine osteoblast-like cell line MC3T3-E1) and the morphological changes under specific periodic traction stress stimulation. The key of bone tissue engineering is to create a cell culture system similar to the physiological environment in vivo. In the past, cell culture in vitro was monolayer, that is, two-dimensional cell culture, which was very different from the living environment of cells in vivo. At present, the establishment of three-dimensional cell culture system is the research focus of bone tissue engineering, and the requirements of different tissue cells for three-dimensional structure carrier are different. In this experiment, the cloned rat skull osteoblast-like cells were selected as seed cells, the three-dimensional structure culture system was established, the cell evolution in the process of cell culture was studied, and the morphological changes of cells were further observed under dynamic conditions, which laid a foundation for further exploring the molecular biological effects and signal transduction mechanism of osteoblasts stimulated by mechanical traction stress, so as to guide the treatment of fracture in clinic. Methods: gelatin sponge (2cmx2cmx0.25cm) was used as three-dimensional culture scaffold of MC3T3-E1. Gelatin sponge was derived from purified pig skin collagen (Gelfoam; Upjohn, Kalamazoo, MI), 100 渭 l per piece of gelatin sponge, and the number of cells was 1.25 脳 10 ~ 5. Biological traction device (Bio-Stretch System; ICCT Technologies, Inc., Canada) includes a programmed pulse timer, controller, bidirectional regulating DC power supply and a set of solenoid conduit with platform. Petri dishes with gelatin sponges are placed in front of each catheter. One end of the gelatin sponge is fixed to the bottom of the dish, and the other end is wrapped in a plastic clip over a metal rod. After power supply, the magnetic field is generated through the current of the coil to attract the metal rod to move in the direction of the coil and stretch the gelatin sponge. After the power is cut off, the gelatin sponge retracts to the original length. The tensile degree of collagen sponge was 5%, the action frequency was 60 cycles / min, and the action time was 15 min / h. Three samples were taken from the traction group and the control group on the 2nd day, 4th day, 6th day, 8th day and 10th day, respectively, and the cells were counted. Each fast gelatin sponge in the control group and the traction group was evenly cut into four parts and fixed with (Bouin's fluid) for 48 hours, and then the sections were made according to the following procedure: dehydration, paraffin embedding: paraffin section 6 渭 m, dewaxing, hematoxylin eosin staining. He staining was used to observe the morphological changes. Two samples t test was carried out by SAS6.12 statistical software.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R329.2
本文编号：2501714
[Abstract]:1 three-dimensional culture of osteoblast-like cells and morphological changes under traction stress objective: to study the three-dimensional culture method of cloned rat skull osteoblast-like cells (colonal murine osteoblast-like cell line MC3T3-E1) and the morphological changes under specific periodic traction stress stimulation. The key of bone tissue engineering is to create a cell culture system similar to the physiological environment in vivo. In the past, cell culture in vitro was monolayer, that is, two-dimensional cell culture, which was very different from the living environment of cells in vivo. At present, the establishment of three-dimensional cell culture system is the research focus of bone tissue engineering, and the requirements of different tissue cells for three-dimensional structure carrier are different. In this experiment, the cloned rat skull osteoblast-like cells were selected as seed cells, the three-dimensional structure culture system was established, the cell evolution in the process of cell culture was studied, and the morphological changes of cells were further observed under dynamic conditions, which laid a foundation for further exploring the molecular biological effects and signal transduction mechanism of osteoblasts stimulated by mechanical traction stress, so as to guide the treatment of fracture in clinic. Methods: gelatin sponge (2cmx2cmx0.25cm) was used as three-dimensional culture scaffold of MC3T3-E1. Gelatin sponge was derived from purified pig skin collagen (Gelfoam; Upjohn, Kalamazoo, MI), 100 渭 l per piece of gelatin sponge, and the number of cells was 1.25 脳 10 ~ 5. Biological traction device (Bio-Stretch System; ICCT Technologies, Inc., Canada) includes a programmed pulse timer, controller, bidirectional regulating DC power supply and a set of solenoid conduit with platform. Petri dishes with gelatin sponges are placed in front of each catheter. One end of the gelatin sponge is fixed to the bottom of the dish, and the other end is wrapped in a plastic clip over a metal rod. After power supply, the magnetic field is generated through the current of the coil to attract the metal rod to move in the direction of the coil and stretch the gelatin sponge. After the power is cut off, the gelatin sponge retracts to the original length. The tensile degree of collagen sponge was 5%, the action frequency was 60 cycles / min, and the action time was 15 min / h. Three samples were taken from the traction group and the control group on the 2nd day, 4th day, 6th day, 8th day and 10th day, respectively, and the cells were counted. Each fast gelatin sponge in the control group and the traction group was evenly cut into four parts and fixed with (Bouin's fluid) for 48 hours, and then the sections were made according to the following procedure: dehydration, paraffin embedding: paraffin section 6 渭 m, dewaxing, hematoxylin eosin staining. He staining was used to observe the morphological changes. Two samples t test was carried out by SAS6.12 statistical software.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R329.2
【参考文献】
相关期刊论文 前1条
1 荆鑫,赵剑,祝云利,潘欣,周维江,吴海山;pcDNA3-EGFP基因转染骨髓间充质干细胞的实验研究[J];徐州医学院学报;2001年06期
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