接枝密度梯度表面的构建及其对细胞迁移行为的调控

发布时间：2018-06-05 03:00

本文选题：聚乙二醇 + 碱性成纤维细胞生长因子　；参考：《浙江大学》2012年博士论文

【摘要】：细胞迁移是细胞的一个重要功能,在组织再生如血管生成、伤口愈合和骨形成过程中起关键作用。其中,血管平滑肌细胞(VSMC)的迁移是治疗心血管疾病的重要问题。本论文制备了不同分子量(MW2kDa、5kDa和10kDa)和接枝质量(0-859ng/cm2)的聚乙二醇(PEG)表面,并采用X射线光电子能谱仪(XPS)和石英晶体微天平(QCM-D)等技术进行了表征。VSMCs黏附和迁移主要是由PEG的接枝质量而非分子量调控。VSMCs在均匀的PEG表面无规迁移。它们的迁移速率随着PEG接枝质量的增加而先增大后减少；当接枝质量为300-500ng/cm2时,细胞迁移速率最快(～24μm/h)。对细胞的黏附强度、细胞骨架排列以及黏附相关基因和蛋白表达水平进行了研究,发现在PEG接枝密度适中的表面,细胞粘附力适当,表现出最高的迁移速率。基于均匀PEG表面密度对细胞迁移的影响规律,采用注射法制备了PEG接枝密度梯度表面。由于PEG的阻粘作用,随着其密度的增加,VSMCs黏附的数量减少,细胞铺展面积变小。梯度表面在一定程度上可以诱导VSMCs沿梯度方向取向和定向移动。在PEG梯度表面构建20μm条带图案,细胞趋化迁移和迁移的方向性得到进一步提高。细胞迁移的方向性和趋化指数分别高达0.7和0.53,有69%的细胞定向迁移至PEG密度低的区域。为了更好地诱导细胞定向迁移,选用了生物活性更强的碱性成纤维细胞生长因子(bFGF)进行研究。通过肝素与bFGF之间特异性的相互作用在表面上固定bFGF分子。制备了一系列均匀的bFGF表面,密度的变化范围为0-295ng/cm2,免疫荧光染色技术证实了bFGF仍保留有生物活性。bFGF的密度对VSMCs的运动性影响显著,却对骨髓间质干细胞(MSCs)和内皮细胞(ECs)的迁移无明显影响。随着bFGF密度的增加,VSMCs的迁移速率先增加随后减小,在83ng/cm2达到最大(-22μm/h)。对细胞黏附、铺展面积、细胞粘附力、细胞骨架排列和迁移相关蛋白的表达水平进行了研究,揭示在不同密度的bFGF表面,细胞的迁移是通过调节细胞内的信号,特别是myosin IIA的表达来调控的。基于上述研究,利用注射法制备了bFGF梯度。在梯度表面,bFGF的密度逐渐增加,从约130ng/cm2增加至300ng/cm2,斜率为17ng/cm2/mm。由于bFGF强的化学诱导性,细胞沿着bFGF梯度定向排列,约有60%的细胞与梯度方向夹角小于30°,约有25%细胞取向角小于10°。此外,bFGF梯度能有效诱导VSMCs的定向迁移,高达60%的细胞倾向于迁移至高密度bFGF的区域,细胞迁移的方向性和趋化性显著提高。仅仅依靠化学梯度的作用,趋化指数和方向性指数分别高达0.4和0.5。本论文成功地制备了有效诱导细胞定向迁移的梯度材料,阐明了材料诱导细胞迁移的规律和机理,为设计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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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