骨性关节炎中软骨细胞力学研究

Research On Chondrocyte Mechanics In Osteoarthritis

沈星宇

11749079

硕士

生物医学工程

唐斌

2019-05-21

2019-06-27

哈尔滨工业大学

深圳

骨性关节炎（OA）的发生发展是一个复杂的过程，在骨性关节炎的发病过程中力学是一个关键的因素。很多研究在探讨骨性关节炎的发病原因时集中在生物化学因素，而对于在发病过程中的力学影响则较少探讨。本研究探讨了软骨细胞的力学相关的三个方面的问题：（1）骨性关节炎中不同形态的软骨细胞的力学。软骨细胞在体外培养时会呈现梭形和扁平形，以往在力学测量时对不同形态细胞并未区分，本研究分别测量了不同形态的软骨细胞的力学性质，发现梭形软骨细胞的弹性模量小于扁平形细胞的弹性模量。细胞弹性模量与细胞骨架排列方式密切相关，梭形细胞细胞骨架排列方向单一，扁平形细胞细胞骨架排列方向各异。细胞骨架排列方式的不同造成了扁平形细胞的弹性模量高于梭形细胞。梭形细胞相对于扁平形细胞有较快的迁移速度。（2）不同钙离子浓度下软骨细胞的力学性质。骨性关节炎中关节滑液中钙离子浓度随着疾病的加深逐渐增加，钙在骨性关节炎的病程中起到重要作用。本研究模拟不同分级的骨性关节炎患者关节滑液中的钙离子浓度，用浓度70μg/mL-150μg/mL的钙离子处理细胞，发现软骨细胞的弹性模量、细胞和细胞外基质作用力随着钙离子浓度增加呈先上升后下降的趋势，通过F-actin荧光染色检测了细胞骨架密度，通过real-time PCR检测了整合素、肌球蛋白、Ⅱ型胶原蛋白、蛋白多糖等的表达，通过细胞划痕检测细胞在不同钙离子浓度时的迁移，发现软骨细胞的迁移能力随着钙离子浓度呈现先下降后上升的趋势。（3）骨性关节炎中软骨细胞的老化力学。在骨性关节炎的修复中，老化细胞的清除对于软骨的修复有重要作用。在骨性关节炎的发病进程中，软骨的硬度随着疾病的加深逐渐增加。本研究探讨了不同基底硬度的软骨细胞老化的影响，经过过氧化氢诱导早发性老化后，发现生长在较硬基底上的软骨细胞老化程度更高，在较软基底上生长的软骨细胞老化程度较低。炎症相关因子MMP13、il1β和老化相关基因CDKN2也随着基底硬度的降低而降低。探明软骨细胞力学行为与其迁移行为之间的可能内在关联，将明确软骨细胞力学行为在OA发病机制中的作用，加深我们对OA病理的认识，从而促进相关OA临床治疗手段的发展。

The development of osteoarthritis (OA) is a complex process, and mechanics is a key factor in the pathogenesis of osteoarthritis. Many studies have focused on biochemical factors in osteoarthritis, but less on the mechanical effects on the pathogenesis. This study explores three aspects of the mechanics related to chondrocytes:(1) The mechanics of different morphologies of chondrocytes in osteoarthritis. Chondrocytes are fusiform-shaped and flat-shaped when cultured in vitro. They have not been distinguished by different morphological cells in mechanical measurements. This study measured the mechanical properties of different morphologies of chondrocytes and found the Young's modulus of fusiform chondrocytes was smaller than that of flat cells. The cell elastic modulus is closely related to the arrangement of the cytoskeleton. The cytoskeleton of the fusiform cell is arranged in a single direction, and the cytoskeleton of the flat cell is arranged in different directions. The different arrangement of the cytoskeleton causes flat cells’ modulus to be higher than fusiform cells. Fusiform cells have a faster migration rate than flat cells.(2) Mechanical properties of chondrocytes at different calcium ion concentrations. Calcium ion concentration in synovial fluid of osteoarthritis increases with the deepening of the disease, and calcium plays an important role in the course of osteoarthritis. This study simulates the calcium concentration in the synovial fluid of patients with different degrees of osteoarthritis. The cells are treated with calcium ions at a concentration of 70ug/mLto 150ug/mL. It was found that the elastic modulus, cell and extracellular matrix forces of chondrocytes increased first and then decreased with the increase of calcium ion concentration. The cytoskeletal density was detected by F-actin fluorescence staining. Expression of myosin, type II collagen, proteoglycan, integrin was detected by real-time PCR. The wound healing assay was used to detect the migration of cells at different calcium ion concentrations, and it was found that the migration ability of chondrocytes increased first and then increased with the concentration of calcium ions.(3) Aging mechanics of chondrocytes in osteoarthritis. In the repair of osteoarthritis, the removal of senescent cells plays an important role in the repair of cartilage. In the pathogenesis of osteoarthritis, the hardness of cartilage gradually increases as the disease progresses. This study investigated the effects of chondrocyte aging on different matrix hardness. After hydrogen peroxide induced premature senescence, it was found that chondrocytes growing on harder substrates were more senescent, and chondrocytes growing on softer substrates were less aging. The inflammation-related factors MMP13, il1β and the aging-related gene CDKN2 also decrease as the hardness of the substrate decreases.To explore the possible intrinsic relationship between the mechanical behavior of chondrocytes and their migration behavior, the role of chondrocyte mechanical behavior in the pathogenesis of OA will be clarified, and our understanding of OA pathology will be deepened, thus promoting the development of relevant OA clinical treatment methods.

骨性关节炎 软骨细胞 形态差异 钙离子 基底硬度 老化细胞

osteoarthritis chondrocytes morphological difference calcium ion substrate stiffness cell senescence

中文

联合培养

南方科技大学

沈星宇. 骨性关节炎中软骨细胞力学研究[D]. 深圳. 哈尔滨工业大学,2019.