IL-1β对人皮肤瘢痕成纤维细胞增殖及胶原蛋白代谢的影响

发布时间：2018-04-26 04:32

  本文选题：增生性瘢痕 + 成纤维细胞　； 参考：《太原理工大学》2015年硕士论文

【摘要】：增生性瘢痕是人体皮肤真皮层受到创伤后引起的一系列真皮层增生。在创伤修复过程中,成纤维细胞起到至关重要的作用,了解和控制成纤维细胞的生物学行为是促进伤口愈合和预防瘢痕形成的基础和关键。近年来,随着现代细胞生物学和分子生物学在瘢痕领域的深入研究和迅速发展,对成纤维细胞、细胞外基质和细胞因子三者之间的相互作用有了进一步的认识。成纤维细胞可以分泌细胞外基质,而成纤维细胞的大量增殖、细胞外基质中胶原合成与降解、部分细胞因子的大量产生以及三者之间的密切关系构成了增生性瘢痕形成的生物学基础。研究发现,在创伤初期,表皮细胞和真皮细胞将分泌白介素-1β(Interleukin-1β, IL-1β),且IL-1p具有促进成纤维细胞、血管内皮细胞增殖和细胞外基质沉积作用。本文实验在细胞水平,并结合生物学的方法和手段,研究了人皮肤成纤维细胞(human skin fibroblasts, HFB)和瘢痕成纤维细胞(hypertrophic scar fibroblasts, HSF)在离体状态下,不同浓度外源性的IL-1β刺激后,其增殖情况和细胞外基质中胶原蛋白代谢的改变,有助于进一步了解瘢痕形成的生物学机制以及为增生性瘢痕的治疗方法及评估提供重要参考。本文的主要工作及结论如下： (1)采用组织块贴壁法提取并培养HSF,采用胶原酶消化法提取并培养HFB, HSF与HFB在形态上无明显差异,均呈长梭形；本实验均使用第3-5代生长状态良好且处于对数增殖期的细胞,以每孔1.5×104个细胞的密度接种于6孔培养板或103个细胞的密度接种于96孔培养板,24h后,分别用2.5、5、10与20ng/mL浓度的IL-1β培养细胞24h,以0ng/mL浓度的IL-1β培养细胞24h为对照组；检测细胞增殖以及细胞外基质中胶原蛋白代谢的相关指标。 (2)采用MTT比色法测定各组细胞的增殖情况,流式细胞分析法测定各组细胞的细胞周期,并分析各组成纤维细胞S期所占比例。上述两种检测方法均得出：IL-1β可以促进HFB增殖,抑制HSF增殖,从而抑制瘢痕的形成；并且随着培养液中IL-1β浓度的升高,其促进HFB的增殖作用越明显,抑制HSF的增殖也越明显。 (3)采用ELISA法检测各组成纤维细胞上清液中Ⅰ型胶原蛋白和Ⅲ型胶原蛋白的浓度。上述检测方法得出：IL-1β可以促进HFB合成并分泌Ⅰ、Ⅲ型胶原蛋白,抑制HSF合成分泌Ⅰ、Ⅲ型胶原蛋白,从而抑制瘢痕发生；并且随着培养液中IL-1β浓度升高,其促进HFB胶原蛋白代谢的作用越明显,抑制HSF胶原蛋白代谢的作用也越明显。IL-1β刺激能够影响成纤维细胞Ⅰ、Ⅲ型胶原蛋白的合成与分泌,且与IL-1β的浓度有关,期望该结论能为相关疾病临床用药剂量控制提供理论依据。
[Abstract]:Hypertrophic scar is a series of cortical hyperplasia caused by trauma of human dermis. Fibroblasts play an important role in wound healing. Understanding and controlling the biological behavior of fibroblasts is the basis and key to promote wound healing and prevent scar formation. In recent years, with the deep research and rapid development of modern cell biology and molecular biology in the field of scar, the interaction among fibroblasts, extracellular matrix and cytokines has been further understood. Fibroblasts secrete extracellular matrix (ECM), and fibroblast proliferation, collagen synthesis and degradation in ECM. The production of some cytokines and their close relationship constitute the biological basis of hypertrophic scar formation. It was found that in the early stage of trauma, the epidermal cells and dermal cells secreted interleukin-1 尾 -interleukin-1 尾, IL-1 尾, and IL-1p promoted fibroblast, vascular endothelial cell proliferation and extracellular matrix deposition. In this paper, at the cell level, combined with biological methods and methods, we studied the effects of exogenous IL-1 尾 on human skin fibroblasts skin fibroblasts, HFB) and scar fibroblasts stimulated by hypertrophic scar fibroblasts, HSF) in vitro. Its proliferation and changes of collagen metabolism in extracellular matrix are helpful to further understand the biological mechanism of scar formation and provide important reference for the treatment and evaluation of hypertrophic scar. The main work and conclusions are as follows: (1) HSF and HFB were extracted and cultured by tissue mass adherence and collagenase digestion, respectively. There was no significant difference in morphology between HSF and HFB, and the cells in good growth state and logarithmic proliferative phase were used in this experiment. The density of 1.5 脳 104 cells per well was inoculated in 6-well culture plate or 103 cell density in 96-well culture plate for 24 hours. The cells were cultured with IL-1 尾 at the concentration of 2.5 渭 g / 10 and 20ng/mL for 24 h, respectively, and IL-1 尾 with 0ng/mL concentration for 24 h as control group. Cell proliferation and collagen metabolism in extracellular matrix were measured. (2) MTT colorimetric assay was used to determine cell proliferation, flow cytometry was used to determine cell cycle, and the percentage of fibroblasts in S phase was analyzed. The above two methods showed that IL-1 尾 could promote the proliferation of HFB, inhibit the proliferation of HSF and inhibit the formation of scar, and with the increase of the concentration of IL-1 尾 in the culture medium, the more obvious the effect of promoting the proliferation of HFB, the more obvious the inhibition of the proliferation of HSF. ELISA assay was used to detect the concentration of collagen type 鈪，

本文编号：1804512