流体剪切力影响人骨髓间充质干细胞增殖与成骨分化的实验研究

发布时间：2018-05-24 20:51

本文选题：流体剪切力 + 人骨髓间充质干细胞　；参考：《第三军医大学》2005年硕士论文

【摘要】： 生物反应器是近年来生物工程方面研究热点之一,广泛应用于培养细胞及组织复合物。灌注式生物反应器不仅具有三维动力性培养的优点,而且其动力性环境可对种子细胞产生流体剪切应力(fluid shear stress,FSS),因此在骨组织工程研究领域呈现出良好的应用前景。骨骼的骨量和正常生理状态的维持有赖于对骨骼施加适当的应力刺激。研究表明骨骼基质形变引起的细胞外液流动所形成的FSS是作用于骨骼细胞的主要应力。FSS对骨骼细胞正常生理活动及增殖与分化有着重要的作用。平行平板流动腔(parallel-plate flow chamber,PPFC)作为一种可以提供较精确液体层流的实验装置,常被用于研究细胞流体力学。骨髓间充质干细胞(mesenchymal stem cells, MSCs)被认为是最有发展前景的骨组织工程种子细胞之一。hMSCs存在于骨髓基质中,其生理环境与骨骼细胞类似,也应是应力敏感细胞,推测FSS作用于hMSCs后应该产生与骨骼细胞相似的生物学效应。实验通过对平行平板流动腔进行改良制作,利用其对hMSCs进行FSS刺激,通过倒置显微镜及透射电镜观察细胞形态学改变,细胞增殖通过MTT法及流式细胞仪检测,成骨分化通过碱性磷酸酶和骨钙素活性测定、碱性磷酸酶染色、钙结节茜素红染色及四环素荧光标记检测。我们通过观察FSS刺激hMSCs体外增殖及向成骨方向分化的作用,为进一步研究FSS刺激hMSCs体外增殖及分化的机制、利用灌注式生物反应器培养组织工程骨提供理论依据。主要研究结果及结论如下: 一、改良的平行平板流动腔的制备及其优点。流动腔以有机玻璃作为流动腔管道部分,塑料薄膜用来密封流动腔和保持流动腔高度,应用塑料细胞培养瓶底作为细胞贴附载体,静脉输液管作为灌流液循环管道,以长尾夹固定和密封平行平板流动腔。整个装置以放射线辐照消毒灭菌。此模型较其它报道的流体剪切力刺激模型制作简单,成本低廉,操作简便。实验证明此系统作为hMSCs流体力学的研究模型是科学、可行的。二、流体剪切力可促进人骨髓间充质干细胞增殖。将传代的第三代hMSCs分为
[Abstract]:Bioreactor is one of the research hotspots in bioengineering in recent years. It is widely used in cultured cells and tissue complexes. Perfusion bioreactor not only has the advantage of three-dimensional dynamic culture, but also its dynamic environment can produce fluid shear stress and shear stress in seed cells, so it has a good application prospect in the field of bone tissue engineering. The maintenance of bone mass and normal physiological state depends on appropriate stress stimulation on bone. The results showed that FSS formed by extracellular fluid flow induced by deformation of bone matrix is the main stress of bone cells. FSs play an important role in normal physiological activities, proliferation and differentiation of bone cells. Parallel-plate flow chamber PPFC (parallel plate flow chamber), as an experimental device which can provide accurate liquid laminar flow, is often used to study cell hydrodynamics. Mesenchymal stem cells, MSCs) is considered to be one of the most promising seed cells for bone tissue engineering. It is speculated that FSS acting on hMSCs should produce biological effects similar to those of skeletal cells. In the experiment, the parallel plate flow chamber was modified to stimulate hMSCs with FSS, the morphological changes were observed by inverted microscope and transmission electron microscope, and cell proliferation was detected by MTT and flow cytometry. Osteogenic differentiation was determined by alkaline phosphatase and osteocalcin activity, alkaline phosphatase staining, calcium nodule alizarin red staining and tetracycline fluorescence labeling. By observing the effects of FSS on the proliferation and osteogenic differentiation of hMSCs in vitro, we provide a theoretical basis for further studying the mechanism of FSS stimulating proliferation and differentiation of hMSCs in vitro and using perfusion bioreactor to culture tissue engineered bone. The main findings and conclusions are as follows: First, the preparation and advantages of the improved parallel plate flow chamber. The flow chamber is filled with plexiglass, the plastic film is used to seal the flow chamber and keep the height of the flow chamber, the bottom of the plastic cell culture bottle is used as the cell attachment carrier, and the intravenous infusion tube is used as the perfusion fluid circulation pipe. The parallel plate flow chamber is fixed and sealed with a long tail clip. The whole device is sterilized by radiation irradiation. Compared with other reported fluid shear stimulation models, this model is simple to make, low cost and easy to operate. The experiment proves that the system is scientific and feasible as the research model of hMSCs hydrodynamics. Second, fluid shear stress can promote the proliferation of human bone marrow mesenchymal stem cells. Divide the third generation of hMSCs into
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329.2

【引证文献】