發(fā)布時(shí)間：2018-05-24 16:56

  本文選題：間充質(zhì)干細(xì)胞 + β-磷酸三鈣　； 參考：《上海交通大學(xué)》2008年碩士論文

【摘要】： 目的:建立一套小規(guī)模MSC高效擴(kuò)增反應(yīng)器系統(tǒng),在實(shí)驗(yàn)室水平快速擴(kuò)增骨髓間充質(zhì)干細(xì)胞,并同期建立成骨分化誘導(dǎo)及動(dòng)力學(xué)研究方法,為臨床骨損傷修復(fù)在短期獲得定量組織、細(xì)胞數(shù)提供實(shí)驗(yàn)室向生產(chǎn)過(guò)渡。 材料方法:(1)采用平行實(shí)驗(yàn)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(xì)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(cè)試中,用廉價(jià)易貼壁的Vero進(jìn)行接種,觀察與2D平板培養(yǎng)相比較的擴(kuò)增速率優(yōu)勢(shì)。(3)在實(shí)驗(yàn)室利用可觀測(cè)雙聯(lián)小室設(shè)計(jì)并搭建體外灌注生物反應(yīng)器。(4)將提取的2代大鼠骨髓間充質(zhì)干細(xì)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(kāi)始連續(xù)灌注培養(yǎng),于定期分別取樣,與二維平板對(duì)照比較,CCK-8染色檢測(cè)細(xì)胞生長(zhǎng)趨勢(shì)、遷徙率變化、ALP活性染色測(cè)試有無(wú)成骨誘導(dǎo)劑下的成骨誘導(dǎo)水平。干燥細(xì)胞-支架混合體,在掃描電鏡下觀察細(xì)胞生長(zhǎng)狀態(tài)、遷移能力和支架親和水平。 結(jié)果:(1)制備出平滑均勻、有一定強(qiáng)度和較低脆碎度、細(xì)胞懸液和培養(yǎng)基吸收高效、孔隙率達(dá)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(kuò)增速率。(3)反應(yīng)器在流速15.7 ml/h左右能優(yōu)效擴(kuò)增間充質(zhì)干細(xì)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(dú)特的成骨誘導(dǎo)能力,并且相對(duì)于平板培養(yǎng),CCK-8染色檢測(cè)證明細(xì)胞擴(kuò)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(dǎo)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(fù)要求。電鏡掃描顯示出胞外基質(zhì)的分泌使細(xì)胞集落與支架很好的親和,并在層面結(jié)構(gòu)顯示出獨(dú)特的生長(zhǎng)適應(yīng)性。 結(jié)論:用優(yōu)化的工藝和處方,可獲得外觀規(guī)則光滑、質(zhì)地均勻堅(jiān)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(xì)胞貼附。采用雙聯(lián)小室配合β-TCP三維支架,可實(shí)現(xiàn)在實(shí)驗(yàn)室搭建三維連續(xù)灌注生物反應(yīng)器,具備接種、取樣、觀察、考察遷徙率生長(zhǎng)速率等功能,相對(duì)于二維平板,β-TCP三維支架可顯著提高間充質(zhì)干細(xì)胞擴(kuò)增速度,材料本身可在接近飽和誘導(dǎo)的水平促進(jìn)間充質(zhì)干細(xì)胞成骨分化。單個(gè)細(xì)胞MSC存在成骨誘導(dǎo)劑時(shí)的分化能力與細(xì)胞比生長(zhǎng)速率無(wú)直接關(guān)聯(lián)。干細(xì)胞能在三維支架上分泌胞外基質(zhì),借以相互接觸并融合在支架表面。三維支架層面結(jié)構(gòu)相對(duì)于條狀和柱狀結(jié)構(gòu)更有利于細(xì)胞的貼附與生長(zhǎng)。
[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(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)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(zhì)干細(xì)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(shū)林,靳立軍,余細(xì)勇,張斌;骨髓間充質(zhì)干細(xì)胞的肌源性誘導(dǎo)分化及轉(zhuǎn)染VEGF基因的表達(dá)[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2004年03期

3 王曉萃;吳金生;付文玉;邢海霞;;誘導(dǎo)大鼠間充質(zhì)干細(xì)胞形成的神經(jīng)元樣細(xì)胞的形態(tài)特征[J];解剖學(xué)報(bào);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(zhì)干細(xì)胞的體外實(shí)驗(yàn)研究[J];口腔醫(yī)學(xué)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(fù)合體負(fù)載骨形成蛋白2用于組織工程骨的構(gòu)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(dá);葉啟彬;;純?chǔ)?磷酸三鈣粉末制備和細(xì)胞相容性研究[J];武警醫(yī)學(xué);2007年02期

7 龐永剛,崔鵬程,陳文弦,曹云新;人骨髓間質(zhì)干細(xì)胞作為骨、軟骨組織工程種子細(xì)胞的實(shí)驗(yàn)研究[J];細(xì)胞與分子免疫學(xué)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(qiáng);雍宜民;四[，

本文編號(hào)：1929846