基于表面引发ATRP的梯度材料构建及其对细胞迁移行为的调控
发布时间：2018-08-09 17:00
【摘要】：细胞迁移在人体生命过程中起着非常重要的作用。它参与了胚胎形成、伤口愈合、免疫反应等生理过程,也与肿瘤转移紧密相关。体内细胞定向迁移是在体内生物、化学或物理的梯度信号作用下发生的。体外构建梯度材料可在避开体内复杂的环境的同时在一定程度上模拟相应的生理过程,从而突出特定因素对细胞迁移的影响、验证生物材料控制迁移行为的可行性,并对细胞迁移行为进行调控。本文利用表面引发原子转移自由基聚合(SI-ATRP)结合注射法制备了三种不同斜率(0.8、1.6、3.2nm/mm)的聚甲基丙烯酸羟乙酯(PHEMA)分子量(厚度)梯度。通过X射线光电子能谱(XPS)和椭圆偏振光谱对梯度的化学组成和厚度变化进行了表征。在PHEMA梯度表面,血管平滑肌细胞(SMC s)粘着斑和肌动蛋白的表达随着PHEMA的厚度(亲水性)增加而减少,细胞粘附数量、铺展面积和粘附力也相应减少。SMCs在梯度表面趋向于沿着与梯度平行的方向排列,并且向PHEMA厚度低的一端迁移,其趋势随梯度斜率的增加而增大。在PHEMA厚度为3 nm时,迁移速率最大,定向迁移趋势最明显。最佳情况下,有87%的细胞在梯度的诱导下定向迁移。体内组织修复涉及多种细胞的迁移,特定细胞的迁移会促进组织修复,而另一些细胞的迁移会导致组织功能异常。针对血管粥样硬化和“再狭窄”这一具体问题,需要选择性地促进内皮细胞(ECs)迁移同时抑制平滑肌细胞(SMCs)的迁移。通过注射-回填法,结果SI-ATRP和点击化学技术,制备了PHEMA/YIGSR(层粘连蛋白衍生多肽)的互补密度梯度,利用荧光标记对梯度中两组分的变化进行了直接表征,并进一步利用XPS和石英晶体微天平(QCM-d)对互补梯度的组成进行了定量分析。PHEMA密度沿着梯度方向线性减小,YIGSR密度相应线性增加,斜率分别为-48.9ng/cm2·mm和80.4 ng/cm2·mm。在互补梯度表面PHEMA和YIGSR的密度分别为193 ng/cm2和308 ng/cm2处,有82%的ECs沿着YIGSR密度升高(PHEMA密度降低)的方向定向迁移,迁移速率达到了18.2μm/h,是在TCPS表面的5倍,明显快于SMCs (9.7μm/h)。YIGSR与内皮细胞表面的67kD层粘连蛋白结合蛋白(67LR)的特异性相互作用是互补梯度选择性促进内皮细胞迁移的主要原因,而PHEMA的存在降低了细胞与表面的非特异性作用,提高了细胞迁移的定向性。针对神经修复中成纤维细胞(FIBs)迁移太快导致神经纤维化这一问题,本文在抑制FIBs迁移的同时选择性地促进了施旺细胞(SCs)的迁移。在聚磺酸甜菜碱(PDMAPS)和KHIFSDDSSEK(神经细胞粘附分子NCAM衍生多肽,与SCs特异作用)的互补密度梯度表面,KHI的密度从0mm处的“0”线性增加到5mm处的797ng/cm2,而PDMAPS则从555 ng/cm2降低到160 ng/cm2。SCs沿着KHI密度升高(PDMAPS密度降低)的方向定向迁移,迁移速率较在未处理的玻片表面翻倍,12 h净迁移距离增加到原来的3倍。FIBs在互补梯度表面的迁移速率仅为其在玻片表面的60%,且没有表现出明显的方向性。综合两者,SCs在互补梯度表面迁移能力优于FIBs。基底表面KHIFSDDSSEK(模拟NCAM的胞外片段)与SCs表面的NCAM具有特异性相互作用：PDMAPS则降低了细胞与表面的非特异性作用,两种因素的协同作用为SCs迁移提供了适当的牵引力,从而特异性地促进其迁移。本文成功通过SI-ATRP制备了结构可控的二维梯度材料,运用阻粘聚合物的梯度提高了细胞迁移的速率和方向性；运用功能多肽特异性地促进目标细胞的迁移。在国际上较先利用前述两者互补梯度的协同作用使得目标细胞迁移的方向性和速度同时提高,为设计更复杂的功能化生物材料提供了新的思路。
[Abstract]:Cell migration plays a very important role in human life. It participates in the physiological processes such as embryo formation, wound healing, immune response and other physiological processes, which are also closely related to tumor metastasis. The directional migration of cells in vivo is produced by the gradient signal of biological, chemical or physical in vivo. In vitro construction of gradient material can avoid the body complex. The mixed environment simulates the physiological process to a certain extent, thus highlights the effect of specific factors on cell migration, verifies the feasibility of controlling migration behavior by biomaterials and regulates the migration behavior of cells. In this paper, three different kinds of different kinds of surface induced atom transfer radical polymerization (SI-ATRP) combined with injection method are used in this paper. The molecular weight (thickness) gradient of the slope (0.8,1.6,3.2nm/mm) of poly (hydroxyethyl methacrylate) (PHEMA). The chemical composition and thickness of the gradient were characterized by X ray photoelectron spectroscopy (XPS) and ellipsometry spectrum. On the PHEMA gradient surface, the expression of adhesion and actin in vascular smooth muscle cells (SMC s) with the thickness of PHEMA (the thickness of the vascular smooth muscle cells (SMC s). Hydrophilicity increased and decreased, the number of cell adhesion, spreading area and adhesive force also correspondingly reduced.SMCs on the gradient surface, which tended to follow the gradient parallel to the gradient, and moved to the lower end of PHEMA. The trend was increased with the gradient of gradient. When the thickness of PHEMA was 3 nm, the migration rate was the largest and the orientation migration trend was the most. Obviously, at best, 87% of the cells migrate under the induction of gradient. In vivo tissue repair involves the migration of a variety of cells, the migration of specific cells will promote tissue repair, and the migration of other cells will lead to abnormal tissue function. The migration of endothelial cells (ECs) was simultaneously inhibited and the migration of smooth muscle cells (SMCs) was inhibited. The complementary density gradient of PHEMA/YIGSR (laminin derived polypeptide) was prepared by SI-ATRP and click chemical technology by injection backfilling, and the changes in the two components in the gradient were directly characterized by fluorescence labeling, and XPS and quartz were further utilized. The crystal microbalance (QCM-d) has a quantitative analysis of the composition of the complementary gradient. The density of.PHEMA decreases linearly along the gradient direction, and the density of YIGSR increases linearly. The slope of the slope is -48.9ng/cm2. Mm and 80.4 ng/cm2 mm. respectively. The density of PHEMA and YIGSR on the complementary gradient surface is 193 ng /cm2 and 308 ng/cm2, and 82% of the densities are along the density. The directional migration of elevated (PHEMA density reduction) was 18.2 mu m/h, 5 times on the TCPS surface, and the specific interaction of 67kD laminin binding protein (67LR) on the surface of SMCs (9.7 mu m/h).YIGSR was the main cause of complementary gradient selectivity in promoting endothelial cell migration, while PHEMA was stored. In order to reduce the migration of FIBs, the migration of FIBs and the migration of Schwann cells (PDMAPS) and KHIFSDDSSEK (PDMAPS and KHIFSDDSSEK) are selectively promoted. The complementary density gradient surface of the nerve cell adhesion molecule NCAM derived polypeptide and SCs specific action, the density of KHI increased from "0" in 0mm to 797ng/cm2 at 5mm, while PDMAPS decreased from 555 ng/cm2 to 160 ng/cm2.SCs along the direction of KHI density increase (PDMAPS density), and the migration rate was less than that of untreated glassy. The net migration distance of the 12 h is increased to the original 3 times of the original 3 times, the migration rate of.FIBs on the complementary gradient surface is only 60% on the surface of the slide, and there is no obvious direction. In the synthesis, the migration ability of SCs on the complementary gradient surface is superior to that of the FIBs. base surface KHIFSDDSSEK (the simulated NCAM's extracellular fragment) and the NCAM implements on the SCs surface. There is a specific interaction: PDMAPS reduces the non specific effect of cell and surface, and the synergistic effect of the two factors provides the appropriate traction for SCs migration, which specifically promotes its migration. In this paper, the structure controlled two-dimensional gradient material was successfully prepared by SI-ATRP, and the cell migration was enhanced by the gradient of the hindrance polymer. The speed and orientation of the migration and the use of functional peptides specifically promote the migration of the target cells. In the world, the synergy between the two complementary gradients is used to improve the direction and speed of the migration of the target cells at the same time, and provides a new idea for the design of more complex functional biomaterials.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.08
，
本文编号：2174729
[Abstract]:Cell migration plays a very important role in human life. It participates in the physiological processes such as embryo formation, wound healing, immune response and other physiological processes, which are also closely related to tumor metastasis. The directional migration of cells in vivo is produced by the gradient signal of biological, chemical or physical in vivo. In vitro construction of gradient material can avoid the body complex. The mixed environment simulates the physiological process to a certain extent, thus highlights the effect of specific factors on cell migration, verifies the feasibility of controlling migration behavior by biomaterials and regulates the migration behavior of cells. In this paper, three different kinds of different kinds of surface induced atom transfer radical polymerization (SI-ATRP) combined with injection method are used in this paper. The molecular weight (thickness) gradient of the slope (0.8,1.6,3.2nm/mm) of poly (hydroxyethyl methacrylate) (PHEMA). The chemical composition and thickness of the gradient were characterized by X ray photoelectron spectroscopy (XPS) and ellipsometry spectrum. On the PHEMA gradient surface, the expression of adhesion and actin in vascular smooth muscle cells (SMC s) with the thickness of PHEMA (the thickness of the vascular smooth muscle cells (SMC s). Hydrophilicity increased and decreased, the number of cell adhesion, spreading area and adhesive force also correspondingly reduced.SMCs on the gradient surface, which tended to follow the gradient parallel to the gradient, and moved to the lower end of PHEMA. The trend was increased with the gradient of gradient. When the thickness of PHEMA was 3 nm, the migration rate was the largest and the orientation migration trend was the most. Obviously, at best, 87% of the cells migrate under the induction of gradient. In vivo tissue repair involves the migration of a variety of cells, the migration of specific cells will promote tissue repair, and the migration of other cells will lead to abnormal tissue function. The migration of endothelial cells (ECs) was simultaneously inhibited and the migration of smooth muscle cells (SMCs) was inhibited. The complementary density gradient of PHEMA/YIGSR (laminin derived polypeptide) was prepared by SI-ATRP and click chemical technology by injection backfilling, and the changes in the two components in the gradient were directly characterized by fluorescence labeling, and XPS and quartz were further utilized. The crystal microbalance (QCM-d) has a quantitative analysis of the composition of the complementary gradient. The density of.PHEMA decreases linearly along the gradient direction, and the density of YIGSR increases linearly. The slope of the slope is -48.9ng/cm2. Mm and 80.4 ng/cm2 mm. respectively. The density of PHEMA and YIGSR on the complementary gradient surface is 193 ng /cm2 and 308 ng/cm2, and 82% of the densities are along the density. The directional migration of elevated (PHEMA density reduction) was 18.2 mu m/h, 5 times on the TCPS surface, and the specific interaction of 67kD laminin binding protein (67LR) on the surface of SMCs (9.7 mu m/h).YIGSR was the main cause of complementary gradient selectivity in promoting endothelial cell migration, while PHEMA was stored. In order to reduce the migration of FIBs, the migration of FIBs and the migration of Schwann cells (PDMAPS) and KHIFSDDSSEK (PDMAPS and KHIFSDDSSEK) are selectively promoted. The complementary density gradient surface of the nerve cell adhesion molecule NCAM derived polypeptide and SCs specific action, the density of KHI increased from "0" in 0mm to 797ng/cm2 at 5mm, while PDMAPS decreased from 555 ng/cm2 to 160 ng/cm2.SCs along the direction of KHI density increase (PDMAPS density), and the migration rate was less than that of untreated glassy. The net migration distance of the 12 h is increased to the original 3 times of the original 3 times, the migration rate of.FIBs on the complementary gradient surface is only 60% on the surface of the slide, and there is no obvious direction. In the synthesis, the migration ability of SCs on the complementary gradient surface is superior to that of the FIBs. base surface KHIFSDDSSEK (the simulated NCAM's extracellular fragment) and the NCAM implements on the SCs surface. There is a specific interaction: PDMAPS reduces the non specific effect of cell and surface, and the synergistic effect of the two factors provides the appropriate traction for SCs migration, which specifically promotes its migration. In this paper, the structure controlled two-dimensional gradient material was successfully prepared by SI-ATRP, and the cell migration was enhanced by the gradient of the hindrance polymer. The speed and orientation of the migration and the use of functional peptides specifically promote the migration of the target cells. In the world, the synergy between the two complementary gradients is used to improve the direction and speed of the migration of the target cells at the same time, and provides a new idea for the design of more complex functional biomaterials.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.08
，
本文编号：2174729
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