基底刚度对创面愈合相关细胞的影响

发布时间：2018-02-04 20:02

本文关键词： 基底刚度创面愈合相关细胞增殖迁移整合素β1　出处：《重庆大学》2011年硕士论文　论文类型：学位论文

【摘要】：在创面愈合阶段,表皮细胞首先迁移至受创真皮,内皮细胞形成新的血管,并用新生毛细血管交联成纤维细胞和巨噬细胞代替由纤维蛋白基质组成的肉芽组织。接着表皮细胞、内皮细胞和成纤维细胞开始增殖和成熟,并最终恢复上皮屏障功能。在创面愈合过程中,创面的刚度发生明显的改变,而力学变化对细胞增殖、迁移和分化有着调控作用。因此,基底刚度的变化对创面愈合相关细胞的影响值得探讨。本文采用人表皮细胞(HACAT)、人脐静脉内皮细胞(HUVEC)和成纤维细胞三种创面愈合相关细胞,以不同刚度的硅胶作为基底,将其接种在硅胶基底上,探索不同刚度基底对创面愈合相关细胞的影响,探索人表皮细胞、内皮细胞与成纤维细胞对外部力学刺激的应激反应,以期为阐明创面愈合机制,促进创面愈合提供可靠的依据和参考意见。本文主要的研究内容和结果为: (1)了解创面愈合过程中刚度的变化,采用不同浓度的硅胶配制不同刚度的硅胶基底以模拟其愈合过程中的刚度变化,观察硅胶基底表面形貌。结果显示以16 kPa、20 kPa、200 kPa来分别模拟正常皮肤的刚度、创面愈合第7天肉芽组织的刚度、创面愈合后期的刚度。扫描电镜观察到不同浓度硅胶基底并未引起表面形貌的差别。 (2)将表皮细胞、内皮细胞和成纤维细胞接种在三种不同刚度的硅胶基底上。通过细胞生长曲线、MTT法和细胞周期检测其增殖情况;用细胞划痕法进行表皮细胞、内皮细胞和成纤维细胞迁移率的测定。发现在刚度高的基底上表皮细胞、内皮细胞和成纤维细胞增殖速度更快,且三种细胞的迁移率远远高于低刚度基底上的细胞。同时发现表皮细胞与成纤维细胞对于基底刚度的变化敏感,而内皮细胞虽然受基底刚度的影响,但其敏感程度不如表皮细胞与成纤维细胞。 (3)应用构建好的整合素β1小RNA干扰载体转染表皮细胞与成纤维细胞,通过瞬时转染,用Western blot检测转染后表皮细胞与成纤维细胞整合素β1蛋白的改变。通过潮霉素持续加压筛选已转染细胞,直到转染细胞株单克隆形成。通过蛋白质印迹发现重组质粒siIntegrinβ1转染表皮细胞与成纤维细胞后,能够抑制细胞中整合素β1的蛋白表达。 (4)将干扰整合β1的表皮细胞和成纤维细胞接种在高刚度的硅胶基底上,以正常表皮细胞与成纤维细胞作为对照。通过细胞生长曲线、MTT法和细胞周期检测其增殖情况;用细胞划痕法进行细胞迁移率的测定。发现在高刚度的基底上干扰整合素β1的表皮细胞和成纤维细胞增殖能力和迁移速度与正常表皮细胞和成纤维细胞相比均有所降低,尤其是迁移速率明显下降。
[Abstract]:At the stage of wound healing, epidermal cells first migrate to the damaged dermis, and endothelial cells form new blood vessels. New capillary crosslinked fibroblasts and macrophages were used to replace granulation tissue composed of fibrin matrix. Then epidermal cells endothelial cells and fibroblasts began to proliferate and mature. And finally restore the function of epithelial barrier. In the process of wound healing, the stiffness of wound changes obviously, while the mechanical changes of cell proliferation, migration and differentiation have a regulatory role. The effect of changes in basal stiffness on wound healing related cells is worth to be explored. Human epidermal cells were used in this paper. Human umbilical vein endothelial cells (HUVECs) and fibroblasts (HUVECs) and fibroblasts (HUVECs) and fibroblasts were inoculated on silica gel substrate with different stiffness silica gel as the substrate. To explore the effects of different stiffness substrates on wound healing related cells, and to explore the stress response of human epidermal cells, endothelial cells and fibroblasts to external mechanical stimulation in order to elucidate the mechanism of wound healing. To promote wound healing to provide reliable basis and reference. The main contents and results of this paper are as follows: 1) to understand the change of stiffness during wound healing, different concentrations of silica gel were used to prepare different stiffness of silica gel substrate to simulate the change of stiffness during the healing process. The surface morphology of silica gel substrate was observed. The results showed that the stiffness of normal skin was simulated by 16kPa20kPa200kPa200, and the stiffness of granulation tissue was observed on the 7th day of wound healing. It was observed by SEM that the surface morphology of different concentrations of silica gel substrate was not different. (2) Epidermal cells, endothelial cells and fibroblasts were inoculated on three kinds of silica gel substrates with different stiffness. The proliferation of epidermal cells, endothelial cells and fibroblasts were detected by MTT assay and cell cycle analysis. The migration rates of epidermal cells, endothelial cells and fibroblasts were measured by cell scratch method. It was found that the proliferation rate of endothelial cells and fibroblasts was faster in the high stiffness basal epidermal cells. Moreover, the mobility of the three cells was much higher than that of the cells on the low stiffness substrate. It was also found that epidermal cells and fibroblasts were sensitive to the change of basal stiffness, while the endothelial cells were affected by the substrate stiffness. But its sensitivity is inferior to that of epidermal cells and fibroblasts. The constructed small RNA interference vector of integrin 尾 1 was used to transfect epidermal cells and fibroblasts by transient transfection. The changes of integrin 尾 1 protein in epidermal cells and fibroblasts after transfection were detected by Western blot, and the transfected cells were screened by hygromycin continuous pressure. The recombinant plasmid siIntegrin 尾 1 was found to be transfected into epidermal cells and fibroblasts by Western blotting. It can inhibit the protein expression of integrin 尾 1 in cells. (4) Epidermal cells and fibroblasts which interfere with integration of 尾 1 were inoculated on a high rigidity silica gel substrate, and normal epidermal cells and fibroblasts were used as control. Cell growth curve was used. The proliferation was detected by MTT and cell cycle. Cell migration was measured by cell scratch method. It was found that the proliferation and migration rate of integrin 尾 1 interfering with the epidermal cells and fibroblasts on the high stiffness substrate were both higher than those of normal epidermal cells and fibroblasts. A little lower. In particular, the migration rate decreased obviously.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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