静电纺丝透明质酸纳米纤维支架及其对细胞迁移行为的影响
发布时间：2019-01-03 10:45
【摘要】：静电纺丝技术制备的纳米纤维在组织工程领域具有十分广泛的作用。但其不利于细胞迁移和渗透的特性限制了它的应用。本课题在纳米纤维中引入透明质酸（HA）—一种具有促进细胞迁移作用的天然胞外基质来解决这个问题。本课题主要研究内容如下： ①本课题通过静电纺丝技术制备了透明质酸（HA）和聚己内酯（PCL）复合纳米纤维，利用天然高分子HA特殊的物理性质和促进细胞迁移的生理功能，改善纳米纤维的局限性。同时，结合人工高分子聚合物PCL优秀的力学性质与稳定性，增加HA的可纺性。以六氟异丙醇（HFIP）和甲酸（FA）分别为溶剂溶解PCL和HA，利用静电纺丝技术制备了PCL、PCL/HA5:1、PCL/HA5:2三种不同的纳米纤维。使用扫描电镜（SEM）、透射电镜（TEM）、傅里叶红外光谱（FTIR）、接触角、拉伸试验等试验对复合纳米纤维进行基础表征表明纳米纤维具有稳定的表面结构和良好的物理性质。 ②在PCL、PCL/HA5:1、PCL/HA5:2三种不同的纳米纤维分别种植人皮肤成纤维细胞FEK4，，观察细胞在材料表面的增殖与胞外基质分泌情况，发现纳米纤维生物相容性良好，可以影响胞外基质的表达。 ③体外利用Transwell和Time-lapse显微镜分别分析HA在纳米纤维中对细胞渗透和迁移的影响，并与HA在2D环境中对细胞迁移能力的影响对比。结果表面HA在2D和3D环境中对细胞迁移有不同的影响。2D环境中，HA通过促进CD44表达，增加细胞丝状伪足的产生，增加细胞迁移的方向性，使得细胞能够更有效的愈合伤口。但是2D环境中，HA减弱了成熟Vinculin的形成，降低了细胞迁移速率。3D环境中，HA促进CD44、Integrin-β1的表达，增加细胞的迁移速率。同时，HA/CD44相互作用可以活化TGF-β1促进MMP-2的表达促进细胞在纳米纤维上的渗透。这些发现有助于我们进一步了解HA在纳米纤维中对迁移的促进作用，为将来应用于组织修复打下基础。 ④将PCL、PCL/HA5:1、PCL/HA5:2三种不同的纳米纤维分别植入SD大鼠皮下，4周后观察发现含HA的支架可以明显促进体内细胞在纳米纤维上的渗透。 本课题研究证明：类似于细胞外基质结构的静电纺丝PCL/HA纳米纤维支架具有稳定的表面结构、理想的物理性质和良好的生物相容性，而且能改善细胞在纳米纤维表面的迁移和渗透。本研究结果为该材料用于组织工程、组织修复、伤口敷料等提供了理论和实验依据。
[Abstract]:Nanofibers prepared by electrostatic spinning technology have been widely used in the field of tissue engineering. However, its application is restricted by its characteristics that are not conducive to cell migration and permeability. In this paper, hyaluronic acid (HA), a natural extracellular matrix, was introduced into nanofibers to solve this problem. The main contents of this paper are as follows: 1 Hyaluronic acid (HA) and polycaprolactone (PCL) nanofibers were prepared by electrostatic spinning. Using the special physical properties of natural polymer HA and the physiological function of promoting cell migration, the limitations of nanofibers were improved. At the same time, the spinnability of HA was improved by combining the excellent mechanical properties and stability of HA. Using hexafluoroisopropanol (HFIP) and formic acid (FA) as solvent to dissolve PCL and HA, three kinds of PCL,PCL/HA5:1,PCL/HA5:2 nanofibers were prepared by electrospinning technique. Scanning electron microscope (SEM), transmission electron microscope (SEM), (TEM), Fourier transform infrared spectroscopy (FTIR),) and tensile test were used to characterize the composite nanofibers. The results show that the nanofibers have stable surface structure and good physical properties. 2Human skin fibroblasts (FEK4,) were implanted with three different kinds of PCL,PCL/HA5:1,PCL/HA5:2 nanofibers to observe the proliferation and extracellular matrix secretion of the cells on the surface of the materials. The results showed that the nanofibers had good biocompatibility. It can affect the expression of extracellular matrix. 3The effects of HA on cell infiltration and migration in nanofibers were analyzed by Transwell and Time-lapse microscope in vitro, and compared with that of HA in 2D environment. Results Surface HA had different effects on cell migration in 2D and 3D environments. In 2D environment, HA could promote the expression of CD44, increase the production of pseudopodia, increase the direction of cell migration, and make the cells heal wounds more effectively. However, in 2D environment, HA decreased the formation of mature Vinculin and decreased the cell migration rate. In 3D environment, HA promoted the expression of CD44,Integrin- 尾 1 and increased the migration rate of cells. At the same time, HA/CD44 interaction can activate TGF- 尾 1 to promote the expression of MMP-2. These findings help us to further understand the role of HA in promoting migration in nanofibers and lay the foundation for future application in tissue repair. (4) three different kinds of PCL,PCL/HA5:1,PCL/HA5:2 nanofibers were implanted into the subcutaneously of SD rats. Four weeks later, it was found that the scaffold containing HA could obviously promote the infiltration of cells on the nanofibers. It is proved that the electrospun PCL/HA nanofiber scaffold, which is similar to the extracellular matrix structure, has stable surface structure, ideal physical properties and good biocompatibility. Moreover, it can improve the migration and penetration of cells on the surface of nanofibers. The results provide theoretical and experimental basis for tissue engineering, tissue repair and wound dressing.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329;TQ340.1;TB383.1
[Abstract]:Nanofibers prepared by electrostatic spinning technology have been widely used in the field of tissue engineering. However, its application is restricted by its characteristics that are not conducive to cell migration and permeability. In this paper, hyaluronic acid (HA), a natural extracellular matrix, was introduced into nanofibers to solve this problem. The main contents of this paper are as follows: 1 Hyaluronic acid (HA) and polycaprolactone (PCL) nanofibers were prepared by electrostatic spinning. Using the special physical properties of natural polymer HA and the physiological function of promoting cell migration, the limitations of nanofibers were improved. At the same time, the spinnability of HA was improved by combining the excellent mechanical properties and stability of HA. Using hexafluoroisopropanol (HFIP) and formic acid (FA) as solvent to dissolve PCL and HA, three kinds of PCL,PCL/HA5:1,PCL/HA5:2 nanofibers were prepared by electrospinning technique. Scanning electron microscope (SEM), transmission electron microscope (SEM), (TEM), Fourier transform infrared spectroscopy (FTIR),) and tensile test were used to characterize the composite nanofibers. The results show that the nanofibers have stable surface structure and good physical properties. 2Human skin fibroblasts (FEK4,) were implanted with three different kinds of PCL,PCL/HA5:1,PCL/HA5:2 nanofibers to observe the proliferation and extracellular matrix secretion of the cells on the surface of the materials. The results showed that the nanofibers had good biocompatibility. It can affect the expression of extracellular matrix. 3The effects of HA on cell infiltration and migration in nanofibers were analyzed by Transwell and Time-lapse microscope in vitro, and compared with that of HA in 2D environment. Results Surface HA had different effects on cell migration in 2D and 3D environments. In 2D environment, HA could promote the expression of CD44, increase the production of pseudopodia, increase the direction of cell migration, and make the cells heal wounds more effectively. However, in 2D environment, HA decreased the formation of mature Vinculin and decreased the cell migration rate. In 3D environment, HA promoted the expression of CD44,Integrin- 尾 1 and increased the migration rate of cells. At the same time, HA/CD44 interaction can activate TGF- 尾 1 to promote the expression of MMP-2. These findings help us to further understand the role of HA in promoting migration in nanofibers and lay the foundation for future application in tissue repair. (4) three different kinds of PCL,PCL/HA5:1,PCL/HA5:2 nanofibers were implanted into the subcutaneously of SD rats. Four weeks later, it was found that the scaffold containing HA could obviously promote the infiltration of cells on the nanofibers. It is proved that the electrospun PCL/HA nanofiber scaffold, which is similar to the extracellular matrix structure, has stable surface structure, ideal physical properties and good biocompatibility. Moreover, it can improve the migration and penetration of cells on the surface of nanofibers. The results provide theoretical and experimental basis for tissue engineering, tissue repair and wound dressing.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329;TQ340.1;TB383.1
【共引文献】
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2 李强,何清义,许建中;VEGF促进骨组织工程血管化的研究进展[J];第三军医大学学报;2005年16期
3 贾建军;王自能;罗新;徐建平;;bFGF及TGF-β1在人胎盘绒毛滋养层的表达[J];广东医学;2006年11期
4 孙梁,胡蕴玉,潘s
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