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
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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
Bioreactor is very hot in the area of bioengineering; it is widely used in culturing cells and tissue. Perfusion bioareactor has advantages in bone tissue engineering, because of the dynamic 3D culture pattern. The dynamic environment can provide fluid shear stress(FSS) on the cultured cells. In order to maintain the mass and the normal functions of the bone, it needs to put suitable stress on the bone. When the interstitial fluid flow was driven by the formation of the bone, it will provide FSS on the cells of the bone. More evidences has supported the fact that FSS is the main stress on the bone cell. And FSS has very important effect on the proliferation and differentiation.
Parallel-plate flow chamber(PPFC) is a experimental facility that can provided FSS of precise magnitude, so it is widely used to study the hydrodynamics of cell. Recently, mensenchymal stem cells(MSCs) is considered one of the most developmental seed cells of bone tissue engineering. Human mesenchymal stem cells is established on the matrix of bone marrow, and the environment is very familiar with bone cells. So we presume that hMSCs is also sensitive to FSS, and the familiar effect can be made when take FSS on hMSCs. In our work, we upgrade the old type of PPFC, and stimulate the hMSCs with FSS using the upgraded PPFC. We observe the form change of hMSCs through inverted microscope and TEM; through MTT and flow cytometer to observe the proliferation; through the ALP Staining, Calcium staining for certain dyestuff, Tetracycline fluorescence labelling, estimating the excretion of osteocalcin by radioimmunoassay and the activity of the cellular ALP to observe the differentiation.
The objective we do the study is to observe the effect of FSS on the proliferation and differentiation of hMSCs. And it can establish the foundation to study the mechanism of the FSS on hMSCs in the future. It also can provide theory and experiment basic for the use of perfusion bioreactor on culturing tissue engineering bone. From the experiment, we get the following result:
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
Bioreactor is very hot in the area of bioengineering; it is widely used in culturing cells and tissue. Perfusion bioareactor has advantages in bone tissue engineering, because of the dynamic 3D culture pattern. The dynamic environment can provide fluid shear stress(FSS) on the cultured cells. In order to maintain the mass and the normal functions of the bone, it needs to put suitable stress on the bone. When the interstitial fluid flow was driven by the formation of the bone, it will provide FSS on the cells of the bone. More evidences has supported the fact that FSS is the main stress on the bone cell. And FSS has very important effect on the proliferation and differentiation.
Parallel-plate flow chamber(PPFC) is a experimental facility that can provided FSS of precise magnitude, so it is widely used to study the hydrodynamics of cell. Recently, mensenchymal stem cells(MSCs) is considered one of the most developmental seed cells of bone tissue engineering. Human mesenchymal stem cells is established on the matrix of bone marrow, and the environment is very familiar with bone cells. So we presume that hMSCs is also sensitive to FSS, and the familiar effect can be made when take FSS on hMSCs. In our work, we upgrade the old type of PPFC, and stimulate the hMSCs with FSS using the upgraded PPFC. We observe the form change of hMSCs through inverted microscope and TEM; through MTT and flow cytometer to observe the proliferation; through the ALP Staining, Calcium staining for certain dyestuff, Tetracycline fluorescence labelling, estimating the excretion of osteocalcin by radioimmunoassay and the activity of the cellular ALP to observe the differentiation.
The objective we do the study is to observe the effect of FSS on the proliferation and differentiation of hMSCs. And it can establish the foundation to study the mechanism of the FSS on hMSCs in the future. It also can provide theory and experiment basic for the use of perfusion bioreactor on culturing tissue engineering bone. From the experiment, we get the following result:
引文
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