王浩 陈群超 殷丽丽

【摘 要】目的：探究不同退变阶段骨关节炎自噬因子ULK1、Beclin1和LC3B在软骨细胞中的表达。方法：根据Kellgren-Lawrence影像学诊断标准，收集正常软骨样品，中期、晚期骨关节炎软骨样品;体外分离、培养软骨细胞，甲苯胺蓝和Ⅱ型胶原免疫组化染色用于鉴定软骨细胞，运用荧光定量PCR及WB技术分别从mRNA及蛋白水平检测ULK1、Beclin1和LC3B的表达。结果：甲苯胺蓝染色显示，软骨细胞核为深蓝色，胞质中有大量异色蓝紫色颗粒。胶原蛋白的免疫荧光染色显示，细胞外基质为棕色，细胞核染为黄褐色。荧光定量PCR及WB结果显示，正常软骨细胞中ULK1、Beclin1、LC3B mRNA及蛋白水平稳定表达，随着骨关节炎程度加重，其表达量逐渐减少，细胞中表达量最低的是晚期骨关节炎组，3组间比较，差异有统计学意义（P < 0.05）。结论：随着骨关节炎的加重，软骨细胞自噬相关因子的表达降低。自噬是维持正常软骨细胞稳态的保护机制，对调控骨关节炎的发展有重要的作用。

【关键词】 骨关节炎;软骨细胞;自噬;退变

Study on the Expression of Autophagy in Osteoarthritis Chondrocyte Degeneration

WANG Hao，CHEN qun-chao，YIN Li-li

【ABSTRACT】Objective：To investigate the expressions of autophagy factors ULK1，Beclin1 and LC3B in chondrocytes of osteoarthritis at different stages of degeneration.Methods：According to the Kellgren-Lawrence imaging diagnostic criteria，normal cartilage samples，mid-term and late osteoarthritis cartilage samples were collected.Chondrocytes were isolated and cultured in vitro;toluidine blue and typeⅡ collagen immunohistochemical staining were used to identify chondrocytes;and the expressions of ULK1，Beclin1 and LC3B were detected by fluorescent quantitative PCR and WB Technology respectively from mRNA and protein levels.Results：Toluidine blue staining showed that the nucleus of cartilage was dark blue，and there were a large number of heterochromatic blue purple granules in the cytoplasm.Immunofluorescence staining of collagen showed that the extracellular matrix was brown and the nucleus was yellowish brown.The results of fluorescence quantitative PCR and WB showed that ULK1，Beclin1 and LC3B were stably expressed in normal chondrocytes at mRNA and protein levels.With the aggravation of osteoarthritis，the expression level gradually decreased.The lowest expression level was in the advanced osteoarthritis group，and the difference was statistically significant

（P < 0.05）.Conclusion：With the aggravation of osteoarthritis，the expression of autophagy related factors in chondrocytes decreased.Autophagy is a protective mechanism to maintain normal chondrocyte homeostasis，and plays an important role in regulating the development of osteoarthritis.

【Keywords】 osteoarthritis;chondroc-yte;autophagy;degeneration

骨關节炎（osteoarthritis，OA）是一种慢性关节疾病，多发生于50岁以上的中老年人，女性发病率高于男性。软骨细胞的减少和软骨基质的降解是OA形成的主要病理变化，其中软骨细胞稳态失衡起着重要的作用[1-2]。在OA的发展过程中，受损的细胞器和降解的大分子物质逐渐积聚在软骨细胞中，破坏了软骨细胞的平衡，细胞合成、分解代谢失衡，导致软骨细胞功能异常，生存能力下降，细胞外基质合成能力下降，从而加速软骨细胞的退变[3]。

自噬（Autophagy）是细胞自我降解并循环利用细胞内组分的过程，在维持细胞环境平衡和提高细胞存活率方面发挥着重要作用[4]。许多疾病的发生和进展都与自噬……