多不饱和脂肪酸微观调控骨关节炎中软骨细胞生物力学特性的机制研究

Osteoarthritis（OA）is a chronic degenerative disease characterized by cartilage degradation and altered chondrocyte phenotype caused by the combined effects of biomechanical and biological factors. The majority of studies have shown that polyunsaturated fatty acids（PUFAs）influence osteoarthritis progression through its biological effects in the inflammatory reaction. However, little is known regarding the biomechanical effects of PUFAs in OA. It has been reported that pro-inflammatory cytokine Interleukin-1 alters the normal volumetric response of chondrocytes to osmotic stress through mechanisms involving F-actin remodeling, the present study therefore tests the hypothesis that PUFAs regulate the biomechanics and volume response of chondrocytes after exposure to inflammatory cytokine and hypo-osmotic stress. Murine chondrocytes were treated with PUFAs and subsequently exposed to inflammatory cytokine and hypo-osmotic stress, temporal changes in cell diameter and the existence of regulatory volume decrease were quantified. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualized in live cells. This study then investigated the possible mechanisms of PUFAs in regulating inflammation reaction, F-actin remodeling and volume regulation through Rho signaling pathway, an animal model was then used to confirm our findings through both in vivo and in vitro experiments. This study will bring new lights to the mechanisms of PUFAs in regulating the biomechanics, volume and inflammatory responses in OA and provide useful information to its clinical and translational research in the future.

骨关节炎（OA）是生物力学与生物学因素失衡导致软骨退化和软骨细胞表型改变的慢性退行性疾病。目前大量研究从生物学角度显示多不饱和脂肪酸可以通过调节炎症反应减缓OA进程，但是对其生物力学作用的研究尚未涉及。有研究显示炎症因子损伤软骨细胞骨架蛋白和低渗透压下的体积调控，本课题假说多不饱和脂肪酸可影响炎性损伤后软骨细胞的生物力学性质和体积调控能力；并拟在炎症因子和低渗透压刺激下，检测多不饱和脂肪酸对软骨细胞体积调节能力的作用，使用微管吸允技术加载力学刺激，计算软骨细胞黏弹性和皮质张力、实时监测活细胞骨架蛋白的动态变化，使用分子生物学技术探索多不饱和脂肪酸调控骨架蛋白和炎症反应信号通路的交叉点，并在OA动物体内验证其生物力学及生物学效应。本课题将从细胞生物力学角度阐明多不饱和脂肪酸调控软骨细胞力学特性、体积调控和炎症反应的机制，为其临床应用转化提供新的思路和实验基础。

骨关节炎（Osteoarthritis, OA）是生物力学与生物学因素失衡导致软骨退化和软骨细胞表型改变的慢性退行性疾病。目前大量研究从生物学角度显示二十碳五烯酸可以通过调节炎症反应减缓OA进程，但是对其生物力学作用的研究尚未涉及。首先，本研究建立体外猪OA软骨细胞模型，RNA-seq结果显示：EPA可通过抑制核糖体合成、细胞周期等通路来减弱IL-1对猪软骨细胞核糖体合成、细胞有丝分裂、DNA结合的促进作用；EPA可通过调节细胞外基质组成来减弱IL-1对猪软骨细胞外基质合成、谷胱甘肽转移酶活性的抑制作用使用。细胞骨架蛋白F-actin免疫组化染色结果显示：EPA显著抑制IL-1升高细胞横截面积、降低细胞圆度和降低猪软骨细胞F-actin荧光强度的作用。其次，本研究建立猪OA软骨组织块模型，GAGs和II型胶原检测显示：EPA显著抑制IL-1引起猪软骨组织块GAGs和II型胶原的丢失；力学测试显示：EPA显著抑制IL-1引起猪软骨组织块样式模量的显著丢失。此外，本研究成功合成复合纳米乳化EPA的玻璃酸钠粘弹性补剂，粒径测量、投射电子显微镜等结果显示：EPA纳米乳液具有良好的稳定性，其粘度和粘弹性表征与原玻璃酸钠注射液相比没有明显差异。综上所述，本研究从体外细胞、软骨组织块实验两个水平证实，EPA可以显著抑制IL-1引起的软骨基质降解和生物力学特性改变，并对其分子生物学机制进行一定程度的揭示。通过构建复合纳米乳化EPA的玻璃酸钠粘弹性补剂，为其今后的临床转化提供了一定的实验基础和支持。