本文選題：脈沖電磁場(chǎng) + 間充質(zhì)干細(xì)胞��； 參考：《第三軍醫(yī)大學(xué)》2005年碩士論文

【摘要】：目的:細(xì)胞是組織構(gòu)建的基礎(chǔ),目前種子細(xì)胞的體外擴(kuò)增需要3周左右時(shí)間,體外構(gòu)建組織工程骨要想滿足臨床需要,如何使種子細(xì)胞大量,快速,優(yōu)質(zhì)擴(kuò)增是面臨的首要問題。本實(shí)驗(yàn)室前期工作證明,特定參數(shù)的脈沖電磁場(chǎng)(pulsed electromagnetic fields,PEMFs)能夠?qū)θ斯撬栝g充質(zhì)干細(xì)胞(human mesenchymal stem cells,hMSCs)的增殖產(chǎn)生促進(jìn)作用,并誘導(dǎo)成骨。然而刺激的頻率不同,是否會(huì)對(duì)細(xì)胞增殖及分化產(chǎn)生不同的影響?在組織工程骨的體外構(gòu)建中我們應(yīng)該選擇怎樣的合適頻率范圍?細(xì)胞是以群體而不是以孤立的形式生長(zhǎng),細(xì)胞間存在物質(zhì)和信息交換,脈沖電磁場(chǎng)是否會(huì)對(duì)縫隙連接細(xì)胞間通訊(Gap junctional intercellular com-munication ,GJ IC )功能產(chǎn)生影響,進(jìn)而影響細(xì)胞增殖分化?這些問題尚缺乏系統(tǒng)的研究。本研究旨在探討不同頻率段PEMFs刺激對(duì)hMSCs體外增殖分化的作用,以期找出適合的頻率范圍,為應(yīng)用PEMFs促進(jìn)hMSCs快速大量?jī)?yōu)質(zhì)擴(kuò)增,誘導(dǎo)成骨分化,研制新型生物反應(yīng)器,以及進(jìn)一步研究PEMFs刺激對(duì)MSCs生物學(xué)效應(yīng)的機(jī)制提供實(shí)驗(yàn)依據(jù)。 方法:1、頻率分組:選擇1-150Hz 頻率范圍,將體外培養(yǎng)的第三代hMSCs 分為5、25、50、75、100、150HzPEMF 刺激組和不施加PEMF 的對(duì)照組。2、參數(shù)選擇:脈沖電磁場(chǎng)刺激強(qiáng)度1.1mT,時(shí)間30min/d,持續(xù)作用21 天。3、倒置相差顯微鏡觀察細(xì)胞形態(tài)。4、四氮噻唑鹽(MTT)比色法檢測(cè)hMSCs 的增殖,繪制細(xì)胞生長(zhǎng)曲線。5、流式細(xì)胞儀檢測(cè)hMSCs 細(xì)胞周期。6、酶化學(xué)法測(cè)定細(xì)胞堿性磷酸酶(ALP)活性。7、放免法測(cè)定細(xì)胞骨鈣素的分泌情況。9、茜素紅鈣染色檢測(cè)鈣結(jié)節(jié)的形成。9、透射電鏡觀察hMSCs 超微結(jié)構(gòu),觀察細(xì)胞間縫隙連接。10、采用熒光光漂白恢復(fù)(Fluorescence redistribution after photobleaching ,FRAP) 技術(shù),通過激光共聚焦顯微鏡檢測(cè)hMSCs 經(jīng)PEMF 刺激后的縫隙連接功能(GJ IC)變化。 結(jié)果:1、經(jīng)PEMF刺激的hMSCs,3d后密度較對(duì)照組增高,體積逐漸增大,形態(tài)變?yōu)槿切�、多角形、鱗形,胞漿中含有較多的基質(zhì)成分和顆粒狀物質(zhì)。不同頻率的PEMF作用組間細(xì)胞形態(tài)無明顯差別。隨培養(yǎng)時(shí)間延長(zhǎng),細(xì)胞逐漸匯合呈鋪路石狀,進(jìn)而出現(xiàn)重疊生長(zhǎng),基質(zhì)堆積,3周后基質(zhì)礦鹽沉積并融合成圓形或卵圓形的鈣化結(jié)節(jié)。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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R35

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 溫磊;脈沖電流刺激對(duì)MSCs與心肌細(xì)胞共培養(yǎng)層分化狀態(tài)的作用研究[D];第三軍醫(yī)大學(xué);2012年

，

本文編號(hào)：1824332