不同频率脉冲电磁场促人骨髓间充质干细胞增殖分化的实验研究

发布时间：2018-04-30 11:12

本文选题：脉冲电磁场 + 间充质干细胞　；参考：《第三军医大学》2005年硕士论文

【摘要】：目的:细胞是组织构建的基础,目前种子细胞的体外扩增需要3周左右时间,体外构建组织工程骨要想满足临床需要,如何使种子细胞大量,快速,优质扩增是面临的首要问题。本实验室前期工作证明,特定参数的脉冲电磁场(pulsed electromagnetic fields,PEMFs)能够对人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)的增殖产生促进作用,并诱导成骨。然而刺激的频率不同,是否会对细胞增殖及分化产生不同的影响?在组织工程骨的体外构建中我们应该选择怎样的合适频率范围?细胞是以群体而不是以孤立的形式生长,细胞间存在物质和信息交换,脉冲电磁场是否会对缝隙连接细胞间通讯(Gap junctional intercellular com-munication ,GJ IC )功能产生影响,进而影响细胞增殖分化?这些问题尚缺乏系统的研究。本研究旨在探讨不同频率段PEMFs刺激对hMSCs体外增殖分化的作用,以期找出适合的频率范围,为应用PEMFs促进hMSCs快速大量优质扩增,诱导成骨分化,研制新型生物反应器,以及进一步研究PEMFs刺激对MSCs生物学效应的机制提供实验依据。方法:1、频率分组:选择1-150Hz 频率范围,将体外培养的第三代hMSCs 分为5、25、50、75、100、150HzPEMF 刺激组和不施加PEMF 的对照组。2、参数选择:脉冲电磁场刺激强度1.1mT,时间30min/d,持续作用21 天。3、倒置相差显微镜观察细胞形态。4、四氮噻唑盐(MTT)比色法检测hMSCs 的增殖,绘制细胞生长曲线。5、流式细胞仪检测hMSCs 细胞周期。6、酶化学法测定细胞碱性磷酸酶(ALP)活性。7、放免法测定细胞骨钙素的分泌情况。9、茜素红钙染色检测钙结节的形成。9、透射电镜观察hMSCs 超微结构,观察细胞间缝隙连接。10、采用荧光光漂白恢复(Fluorescence redistribution after photobleaching ,FRAP) 技术,通过激光共聚焦显微镜检测hMSCs 经PEMF 刺激后的缝隙连接功能(GJ IC)变化。结果:1、经PEMF刺激的hMSCs,3d后密度较对照组增高,体积逐渐增大,形态变为三角形、多角形、鳞形,胞浆中含有较多的基质成分和颗粒状物质。不同频率的PEMF作用组间细胞形态无明显差别。随培养时间延长,细胞逐渐汇合呈铺路石状,进而出现重叠生长,基质堆积,3周后基质矿盐沉积并融合成圆形或卵圆形的钙化结节。2、MTT
[Abstract]:Objective: cells are the basis of tissue construction. At present, it takes about 3 weeks for seed cells to expand in vitro. In order to meet the clinical needs, how to make seed cells large and fast is necessary to construct tissue engineering bone in vitro. High quality amplification is the most important problem. Our previous work has proved that pulsed electromagnetic fields with specific parameters can promote the proliferation of human bone marrow mesenchymal stem cells (BMSCs) and induce osteogenesis. However, the different frequency of stimulation will have different effects on cell proliferation and differentiation. What is the appropriate frequency range for the in vitro construction of tissue engineered bone? Cells grow in colony rather than in isolation. There is material and information exchange between cells. Does pulsed electromagnetic field affect gap junctional intercellular com-munication IC function of gap junction cells, and then affect cell proliferation and differentiation? These problems are still lack of systematic research. The purpose of this study was to investigate the effects of PEMFs stimulation at different frequencies on the proliferation and differentiation of hMSCs in vitro, in order to find out the appropriate frequency range, and to develop a new bioreactor for the application of PEMFs to promote the rapid and high quality expansion of hMSCs and to induce osteogenic differentiation. And to further study the mechanism of PEMFs stimulation on the biological effects of MSCs provide experimental basis. Method: 1, frequency grouping: select 1-150Hz frequency range, The third generation of hMSCs cultured in vitro was divided into 5o 2550N 75100150 Hz PEMF stimulation group and control group without PEMF. The parameters were selected as follows: pulse electromagnetic field stimulation intensity 1.1 Mt, time 30 min / d, continuous action 21 days .3, inverted phase contrast microscope to observe cell morphology .4,thiazolium tetrazolium salt. The proliferation of hMSCs was detected by MTT colorimetry. The cell growth curve was drawn. The cell cycle of hMSCs was detected by flow cytometry. The activity of alkaline phosphatase (ALP) was determined by enzyme chemistry. The secretion of osteocalcin was measured by radioimmunoassay. The formation of calcium nodules was detected by alizarin red calcium staining. The ultrastructure of hMSCs was observed by transmission electron microscope. The gap junction between cells. 10 was observed. The gap junction function of hMSCs stimulated by PEMF was detected by fluorescence bleaching and fluorescence recovery redistribution after photobleaching (FRAP) technique. Results the density and volume of PEMF stimulated hMSCs1 were higher than those of the control group for 3 days, and the shape became triangular, polygonal, scaly, and the cytoplasm contained more matrix and granular substances. There was no significant difference in cell morphology between groups treated with different frequencies of PEMF. With the extension of culture time, the cells gradually converge into paving stone, and then appear overlapping growth. After 3 weeks of matrix accumulation, the mineral salt of the matrix is deposited and fused into a round or oval calcified node .2MTT.
【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】：R35

【引证文献】