本文選題：聚乙二醇 + 堿性成纖維細胞生長因子　； 參考：《浙江大學》2012年博士論文

【摘要】：細胞遷移是細胞的一個重要功能,在組織再生如血管生成、傷口愈合和骨形成過程中起關鍵作用。其中,血管平滑肌細胞(VSMC)的遷移是治療心血管疾病的重要問題。 本論文制備了不同分子量(MW2kDa、5kDa和10kDa)和接枝質(zhì)量(0-859ng/cm2)的聚乙二醇(PEG)表面,并采用X射線光電子能譜儀(XPS)和石英晶體微天平(QCM-D)等技術進行了表征。VSMCs黏附和遷移主要是由PEG的接枝質(zhì)量而非分子量調(diào)控。VSMCs在均勻的PEG表面無規(guī)遷移。它們的遷移速率隨著PEG接枝質(zhì)量的增加而先增大后減少；當接枝質(zhì)量為300-500ng/cm2時,細胞遷移速率最快(～24μm/h)。對細胞的黏附強度、細胞骨架排列以及黏附相關基因和蛋白表達水平進行了研究,發(fā)現(xiàn)在PEG接枝密度適中的表面,細胞粘附力適當,表現(xiàn)出最高的遷移速率。 基于均勻PEG表面密度對細胞遷移的影響規(guī)律,采用注射法制備了PEG接枝密度梯度表面。由于PEG的阻粘作用,隨著其密度的增加,VSMCs黏附的數(shù)量減少,細胞鋪展面積變小。梯度表面在一定程度上可以誘導VSMCs沿梯度方向取向和定向移動。在PEG梯度表面構建20μm條帶圖案,細胞趨化遷移和遷移的方向性得到進一步提高。細胞遷移的方向性和趨化指數(shù)分別高達0.7和0.53,有69%的細胞定向遷移至PEG密度低的區(qū)域。 為了更好地誘導細胞定向遷移,選用了生物活性更強的堿性成纖維細胞生長因子(bFGF)進行研究。通過肝素與bFGF之間特異性的相互作用在表面上固定bFGF分子。制備了一系列均勻的bFGF表面,密度的變化范圍為0-295ng/cm2,免疫熒光染色技術證實了bFGF仍保留有生物活性。bFGF的密度對VSMCs的運動性影響顯著,卻對骨髓間質(zhì)干細胞(MSCs)和內(nèi)皮細胞(ECs)的遷移無明顯影響。隨著bFGF密度的增加,VSMCs的遷移速率先增加隨后減小,在83ng/cm2達到最大(-22μm/h)。對細胞黏附、鋪展面積、細胞粘附力、細胞骨架排列和遷移相關蛋白的表達水平進行了研究,揭示在不同密度的bFGF表面,細胞的遷移是通過調(diào)節(jié)細胞內(nèi)的信號,特別是myosin IIA的表達來調(diào)控的。 基于上述研究,利用注射法制備了bFGF梯度。在梯度表面,bFGF的密度逐漸增加,從約130ng/cm2增加至300ng/cm2,斜率為17ng/cm2/mm。由于bFGF強的化學誘導性,細胞沿著bFGF梯度定向排列,約有60%的細胞與梯度方向夾角小于30°,約有25%細胞取向角小于10°。此外,bFGF梯度能有效誘導VSMCs的定向遷移,高達60%的細胞傾向于遷移至高密度bFGF的區(qū)域,細胞遷移的方向性和趨化性顯著提高。僅僅依靠化學梯度的作用,趨化指數(shù)和方向性指數(shù)分別高達0.4和0.5。 本論文成功地制備了有效誘導細胞定向遷移的梯度材料,闡明了材料誘導細胞遷移的規(guī)律和機理,為設計性能更好的生物材料提供新的思路。
[Abstract]:Cell migration is an important function of cell, which plays a key role in tissue regeneration, such as angiogenesis, wound healing and bone formation. The migration of vascular smooth muscle cells (VSMC) is an important problem in the treatment of cardiovascular diseases.
In this paper, the surface of polyethylene glycol (PEG) with different molecular weights (MW2kDa, 5kDa and 10kDa) and grafting mass (0-859ng/cm2) was prepared, and the X ray photoelectron spectroscopy (XPS) and quartz crystal microbalance (QCM-D) were used to characterize the.VSMCs adhesion and migration of the grafting mass by PEG, while the.VSMCs in the PEG table was not controlled by the molecular weight. The migration rate of the surfaces increased first and then decreased with the increase of the grafting mass of PEG. When the graft mass was 300-500ng/cm2, the cell migration rate was the fastest (~ 24 u m/h). The adhesion strength, the cytoskeleton arrangement, the adhesion related genes and protein expression levels were studied. It was found that the grafting density of PEG was moderate. On the surface, the cell adhesion force is appropriate, showing the highest migration rate.
Based on the influence of surface density of uniform PEG on cell migration, PEG graft density gradient surface was prepared by injection method. Because of the increasing density of PEG, the number of VSMCs adhesion decreased and the cell spreading area became smaller. The gradient surface could induce the orientation and orientation of VSMCs along the gradient direction to a certain extent. On the PEG gradient surface, 20 micron m strips were constructed, and the orientation of cell chemotaxis and migration was further improved. The direction and chemotaxis index of cell migration were as high as 0.7 and 0.53 respectively, and 69% of the cells migrated to the region with low PEG density.
In order to better induce cell oriented migration, a more bioactive basic fibroblast growth factor (bFGF) was selected. A series of homogeneous bFGF surfaces were prepared through the specific interaction between heparin and bFGF. The range of density change was 0-295ng/cm2, and the immunofluorescence staining technique was used. It was confirmed that the density of bFGF still retained bioactive.BFGF had significant influence on the movement of VSMCs, but had no significant effect on the migration of bone marrow mesenchymal stem cells (MSCs) and endothelial cells (ECs). With the increase of bFGF density, the migration rate of VSMCs increased first and then decreased, and reached the maximum in 83ng/cm2 (-22 micron m/h). Cell adhesion, spreading area, and cells were found in 83ng/cm2. The expression level of adhesion, cytoskeleton arrangement and migration related proteins was studied. It was revealed that the cell migration was regulated by regulating the intracellular signal, especially the expression of myosin IIA, on the surface of different density of bFGF.
Based on the above study, the bFGF gradient was prepared by the injection method. On the gradient surface, the density of bFGF increased gradually from about 130ng/cm2 to 300ng/cm2, and the slope was 17ng/cm2/mm. due to the chemical inducibility of bFGF strong, the cells were aligned along the bFGF gradient, about 60% of the cells were less than 30 degrees in the ladder direction, and about 25% of the cells were less than 1. In addition, the bFGF gradient can effectively induce the directional migration of VSMCs, and up to 60% of the cells tend to migrate to the region of high density bFGF, and the direction and chemotaxis of cell migration are significantly improved. The chemotaxis index and the directional index are up to 0.4 and 0.5., respectively, by the chemical gradient.
In this paper, the gradient materials that effectively induce cell oriented migration are successfully prepared, and the rules and mechanisms of material induced cell migration are clarified, which provides a new way of thinking for the design of better biological materials.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R329

【參考文獻】

相關期刊論文 前1條

1 梁光波,張國平,金惠銘,錢睿哲;粘著斑激酶在bFGF引起細胞遷移中的動態(tài)變化及意義[J];生理學報;2004年04期

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本文編號：1980151