本文選題：間充質干細胞 + β-磷酸三鈣��； 參考：《上海交通大學》2008年碩士論文

【摘要】： 目的:建立一套小規(guī)模MSC高效擴增反應器系統(tǒng),在實驗室水平快速擴增骨髓間充質干細胞,并同期建立成骨分化誘導及動力學研究方法,為臨床骨損傷修復在短期獲得定量組織、細胞數(shù)提供實驗室向生產(chǎn)過渡。 材料方法:(1)采用平行實驗探索最佳的β-TCP支架制備處方和工藝,獲得脆碎度、硬度、外觀規(guī)則度、細胞懸液和培養(yǎng)基吸收效率最理想的片狀支架。(2)在靜態(tài)培養(yǎng)測試中,用廉價易貼壁的Vero進行接種,觀察與2D平板培養(yǎng)相比較的擴增速率優(yōu)勢。(3)在實驗室利用可觀測雙聯(lián)小室設計并搭建體外灌注生物反應器。(4)將提取的2代大鼠骨髓間充質干細胞接種于平置支架材料上,先于二氧化碳培養(yǎng)箱中靜置,完全貼附后開始連續(xù)灌注培養(yǎng),于定期分別取樣,與二維平板對照比較,CCK-8染色檢測細胞生長趨勢、遷徙率變化、ALP活性染色測試有無成骨誘導劑下的成骨誘導水平。干燥細胞-支架混合體,在掃描電鏡下觀察細胞生長狀態(tài)、遷移能力和支架親和水平。 結果:(1)制備出平滑均勻、有一定強度和較低脆碎度、細胞懸液和培養(yǎng)基吸收高效、孔隙率達78%的β-TCP支架。(2)在3D靜態(tài)培養(yǎng)比較2D培養(yǎng)中顯示出后期更大的承載量和擴增速率。(3)反應器在流速15.7 ml/h左右能優(yōu)效擴增間充質干細胞。(4)提取的大鼠MSCs在三維支架的灌注培養(yǎng)中,顯示了空白支架獨特的成骨誘導能力,并且相對于平板培養(yǎng),CCK-8染色檢測證明細胞擴增速率提高了約6-10倍,總體堿性磷酸酶活性大大提升,存在成骨誘導劑環(huán)境下,比ALP活性基本保持不變。此外,三維灌注條件下早期呈現(xiàn)整體遷徙率為主,晚期則以局部遷徙為主,其遷徙能力可滿足骨科修復要求。電鏡掃描顯示出胞外基質的分泌使細胞集落與支架很好的親和,并在層面結構顯示出獨特的生長適應性。 結論:用優(yōu)化的工藝和處方,可獲得外觀規(guī)則光滑、質地均勻堅韌、脆碎度低、耐浸泡、高生物親和性的β-TCP三維支架,可供貼壁細胞貼附。采用雙聯(lián)小室配合β-TCP三維支架,可實現(xiàn)在實驗室搭建三維連續(xù)灌注生物反應器,具備接種、取樣、觀察、考察遷徙率生長速率等功能,相對于二維平板,β-TCP三維支架可顯著提高間充質干細胞擴增速度,材料本身可在接近飽和誘導的水平促進間充質干細胞成骨分化。單個細胞MSC存在成骨誘導劑時的分化能力與細胞比生長速率無直接關聯(lián)。干細胞能在三維支架上分泌胞外基質,借以相互接觸并融合在支架表面。三維支架層面結構相對于條狀和柱狀結構更有利于細胞的貼附與生長。
[Abstract]:Objective: to establish a small scale MSC high efficiency amplification reactor system, to rapidly expand bone marrow mesenchymal stem cells (BMSCs) at laboratory level, and to establish a method of osteogenic differentiation induction and kinetics study at the same time. For clinical bone injury repair in a short period of time to obtain quantitative tissue, the number of cells to provide laboratory transition to production. Materials: (1) using parallel experiments to explore the best preparation prescription and process of 尾 -TCP scaffolds, the flake scaffolds with the best degree of brittleness, hardness, regularity of appearance, cell suspension and absorption efficiency of culture medium were obtained in the static culture test. Inoculated with cheap, easily adherent Vero, To observe the advantage of amplification rate compared with 2D flat plate culture, we designed and built an in vitro perfusion bioreactor in the laboratory with observable double chamber, and inoculated the 2 generation rat bone marrow mesenchymal stem cells on the flat scaffold material. The cell growth trend was detected by CCK-8 staining in comparison with two-dimensional plate control after continuous perfusion culture was carried out after complete attachment in a carbon dioxide incubator, and then samples were taken separately at regular intervals, and compared with two-dimensional plate control, CCK-8 staining was used to detect the growth trend of the cells. The change of migration rate and ALP activity staining were used to test the osteogenic induction level with or without osteogenic inducer. The dry cell-scaffold mixture was used to observe cell growth, migration ability and scaffold affinity under scanning electron microscope. Results 1) smooth and uniform, with a certain strength and low brittle degree, cell suspension and medium absorption efficiency, 尾 -TCP scaffold with a porosity of 78%.) compared with 2D culture in 3D static culture, the 尾 -TCP scaffold showed higher loading capacity and amplification rate in 3D static culture than 2D culture.) the MSCs extracted from rat MSCs in 3D static culture could be effectively amplified by expanding mesenchymal stem cells (MSCs) at a flow rate of about 15.7 ml/h. In the perfusion culture of scaffolds, The results showed that the blank scaffold had unique osteoblast induction ability, and compared with plate culture, CCK-8 staining showed that the rate of cell expansion was increased by 6-10 times, the activity of alkaline phosphatase was greatly increased, and the osteoblast inducer was found in the presence of osteogenic inducer. The activity of specific ALP remained basically unchanged. In addition, the whole migration rate was dominant in the early stage and the local migration in the late stage under the condition of 3D perfusion, and its migration capacity could meet the requirements of orthopedic repair. Scanning electron microscopy showed that the secretion of extracellular matrix (ECM) made the cell colony compatible with the scaffold and showed unique growth adaptability in the layer structure. Conclusion: 尾 -TCP scaffolds with smooth appearance, uniform and tough texture, low embrittlement, high biocompatibility and immersion resistance can be obtained by optimizing the process and prescription, and can be attached to adherent cells. Using the double chamber and 尾 -TCP three-dimensional scaffold, the three-dimensional continuous perfusion bioreactor can be built in the laboratory, which has the functions of inoculation, sampling, observation, investigation of the growth rate of migration rate and so on. Compared with two-dimensional plate, 尾 -TCP three-dimensional scaffold can significantly improve the expansion rate of mesenchymal stem cells, and the material itself can promote the osteogenic differentiation of mesenchymal stem cells near saturation level. There was no direct correlation between the differentiation ability of single cell MSC and the specific growth rate of cells in the presence of osteogenic inducer. Stem cells can secrete extracellular matrix on three-dimensional scaffolds to contact each other and fuse on the scaffold surface. Three-dimensional scaffold structure is more favorable for cell adhesion and growth than stripe and columnar structure.
【學位授予單位】：上海交通大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329

【參考文獻】

相關期刊論文 前10條

1 陶欣榮;李文林;蘇娟;王新民;李建秀;胡以平;;人骨髓間充質干細胞在新生小鼠腦中的植入和分化[J];癌變.畸變.突變;2007年01期

2 盛小剛,馮建章,吳書林,靳立軍,余細勇,張斌;骨髓間充質干細胞的肌源性誘導分化及轉染VEGF基因的表達[J];第一軍醫(yī)大學學報;2004年03期

3 王曉萃;吳金生;付文玉;邢海霞;;誘導大鼠間充質干細胞形成的神經(jīng)元樣細胞的形態(tài)特征[J];解剖學報;2007年01期

4 曹穎光;王戎;宋珂;王華均;;TGF-β1、EGFP雙基因修飾骨髓間充質干細胞的體外實驗研究[J];口腔醫(yī)學研究;2007年01期

5 張芳;李衛(wèi)星;;殼聚糖-明膠-磷酸三鈣多孔復合體負載骨形成蛋白2用于組織工程骨的構建[J];山西醫(yī)藥雜志;2007年06期

6 趙斌;呂雙紅;段翠密;匡正達;葉啟彬;;純β-磷酸三鈣粉末制備和細胞相容性研究[J];武警醫(yī)學;2007年02期

7 龐永剛,崔鵬程,陳文弦,曹云新;人骨髓間質干細胞作為骨、軟骨組織工程種子細胞的實驗研究[J];細胞與分子免疫學雜志;2004年03期

8 王居勇;張懷華;沈慧良;麻生義則;陳佳妮;張慶明;李旗;王居強;雍宜民;四[，

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