基于表面引發(fā)ATRP的梯度材料構建及其對細胞遷移行為的調(diào)控
發(fā)布時間：2018-08-09 17:00
【摘要】：細胞遷移在人體生命過程中起著非常重要的作用。它參與了胚胎形成、傷口愈合、免疫反應等生理過程,也與腫瘤轉移緊密相關。體內(nèi)細胞定向遷移是在體內(nèi)生物、化學或物理的梯度信號作用下發(fā)生的。體外構建梯度材料可在避開體內(nèi)復雜的環(huán)境的同時在一定程度上模擬相應的生理過程,從而突出特定因素對細胞遷移的影響、驗證生物材料控制遷移行為的可行性,并對細胞遷移行為進行調(diào)控。本文利用表面引發(fā)原子轉移自由基聚合(SI-ATRP)結合注射法制備了三種不同斜率(0.8、1.6、3.2nm/mm)的聚甲基丙烯酸羥乙酯(PHEMA)分子量(厚度)梯度。通過X射線光電子能譜(XPS)和橢圓偏振光譜對梯度的化學組成和厚度變化進行了表征。在PHEMA梯度表面,血管平滑肌細胞(SMC s)粘著斑和肌動蛋白的表達隨著PHEMA的厚度(親水性)增加而減少,細胞粘附數(shù)量、鋪展面積和粘附力也相應減少。SMCs在梯度表面趨向于沿著與梯度平行的方向排列,并且向PHEMA厚度低的一端遷移,其趨勢隨梯度斜率的增加而增大。在PHEMA厚度為3 nm時,遷移速率最大,定向遷移趨勢最明顯。最佳情況下,有87%的細胞在梯度的誘導下定向遷移。體內(nèi)組織修復涉及多種細胞的遷移,特定細胞的遷移會促進組織修復,而另一些細胞的遷移會導致組織功能異常。針對血管粥樣硬化和“再狹窄”這一具體問題,需要選擇性地促進內(nèi)皮細胞(ECs)遷移同時抑制平滑肌細胞(SMCs)的遷移。通過注射-回填法,結果SI-ATRP和點擊化學技術,制備了PHEMA/YIGSR(層粘連蛋白衍生多肽)的互補密度梯度,利用熒光標記對梯度中兩組分的變化進行了直接表征,并進一步利用XPS和石英晶體微天平(QCM-d)對互補梯度的組成進行了定量分析。PHEMA密度沿著梯度方向線性減小,YIGSR密度相應線性增加,斜率分別為-48.9ng/cm2·mm和80.4 ng/cm2·mm。在互補梯度表面PHEMA和YIGSR的密度分別為193 ng/cm2和308 ng/cm2處,有82%的ECs沿著YIGSR密度升高(PHEMA密度降低)的方向定向遷移,遷移速率達到了18.2μm/h,是在TCPS表面的5倍,明顯快于SMCs (9.7μm/h)。YIGSR與內(nèi)皮細胞表面的67kD層粘連蛋白結合蛋白(67LR)的特異性相互作用是互補梯度選擇性促進內(nèi)皮細胞遷移的主要原因,而PHEMA的存在降低了細胞與表面的非特異性作用,提高了細胞遷移的定向性。針對神經(jīng)修復中成纖維細胞(FIBs)遷移太快導致神經(jīng)纖維化這一問題,本文在抑制FIBs遷移的同時選擇性地促進了施旺細胞(SCs)的遷移。在聚磺酸甜菜堿(PDMAPS)和KHIFSDDSSEK(神經(jīng)細胞粘附分子NCAM衍生多肽,與SCs特異作用)的互補密度梯度表面,KHI的密度從0mm處的“0”線性增加到5mm處的797ng/cm2,而PDMAPS則從555 ng/cm2降低到160 ng/cm2。SCs沿著KHI密度升高(PDMAPS密度降低)的方向定向遷移,遷移速率較在未處理的玻片表面翻倍,12 h凈遷移距離增加到原來的3倍。FIBs在互補梯度表面的遷移速率僅為其在玻片表面的60%,且沒有表現(xiàn)出明顯的方向性。綜合兩者,SCs在互補梯度表面遷移能力優(yōu)于FIBs�；妆砻鍷HIFSDDSSEK(模擬NCAM的胞外片段)與SCs表面的NCAM具有特異性相互作用：PDMAPS則降低了細胞與表面的非特異性作用,兩種因素的協(xié)同作用為SCs遷移提供了適當?shù)臓恳?從而特異性地促進其遷移。本文成功通過SI-ATRP制備了結構可控的二維梯度材料,運用阻粘聚合物的梯度提高了細胞遷移的速率和方向性；運用功能多肽特異性地促進目標細胞的遷移。在國際上較先利用前述兩者互補梯度的協(xié)同作用使得目標細胞遷移的方向性和速度同時提高,為設計更復雜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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R318.08
，
本文編號：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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R318.08
，
本文編號：2174729
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