Analyses of induction of autophagic response related to the chondrocyte cell-death in osteroarthritis.

项目一MIA和ACLT关节内注射诱导的大鼠骨性关节炎与自噬的关系分析：MIA注射后的软骨细胞超微结构显示12h出现空泡，2周时细胞器丢失，出现丰富的空泡。Western blotting(WB)分析显示，MIA注射后12小时软骨组织中LC3 II的表达显著增加，而ATG5-ATG12的表达在MIA注射后2周开始增加。方案II.大鼠生长板软骨细胞自噬分析：生长板软骨细胞超微结构显示有自噬空泡存在，4周龄时尤为明显。WB分析显示，在生长突发期(2w)，Lc3Ⅱ型显著增加，在次级骨化中心发育(6w，2w)时，ATG5-ATg12型显著增加。自噬相关基因在2w的生长板中的表达明显增加。项目III。人骨关节炎软骨细胞的自噬和内质网应激分析：3级软骨细胞的超微结构显示存在丰富的空泡。WB分析显示，三年级学生的学习成绩有显著提高。Lc3、ATG5和ATG7在骨性关节炎3级软骨中的表达水平均高于正常对照组。CHOP和Proceded-XBPI在3级的表达较高。经抗Ub抗体免疫组织化学染色后，骨性关节炎软骨细胞呈强阳性。这些结果表明，退化的软骨细胞表现出自噬和内质网应激的诱导。根据我们的结果，我们认为软骨细胞自噬反应的诱导与软骨组织和生长板的退化有关。

Project I. Analyses of relationship between autophagy and osteroarthritis induced by intra-articular injection of MIA and ACLT in rats : Ultrastructure of a chondrocyte after MIA injection indicated appearance of vacuoles at 12 hours and the loss of organelles and presence of abundant vacuoles at 2 weeks (2w). Western blotting (WB) analyses demonstrated a significant increase in LC3 II form at 12 hours after MIA injection and an increase in Atg5-Atg12 form from 2w in cartilage after MIA injection. On the other hand, an increase of LC3 II form and Atg5-Atg12 form in cartilage extracts of the osteoarthritis induced by ACLT were little.Project II. Analyses of autophagy in chondrocytes of the rat epiphyseal growth plate : Ultrastructure of a chondrocyte in the growth plate showed presence of autophagic vacuoles, especially at 4 weeks-ages. WB analyses demonstrated a significant increase in LC3 II form at growth spurt (2w) and an increase in Atg5-Atg12 form at the development of secondary ossification center, respectively (6w, 2w). The expressions of autophagy-related genes showed significant increase in growth plate at 2w.Project III. Analyses of autophagy and ER-stress in chondrocytes of the human osteoarthritis : Ultrastructure of a chondrocyte in the Grade-3 showed presence of abundant vacuoles. WB analyses demonstrated a significant increase in LC3 II form in Grade-3. The expression levels of LC3, ATG5 and ATG7, was higher in Grade-3 cartilages of the osteoarthritis than in those of the controls. The expression of CHOP and processed-XBPI was higher in Grade-3. After immunohistochemical staining with anti-Ub antibody, the chondrocytes in the osteoarthritis were strongly stained. These results indicate that the degenerated chondrocytes demonstrate induction of autophagy and the endoplasmic reticulum stress. On the basis of our results, we propose that induction of autophagic response in chondrocytes has relevance to degeneration of cartilage tissues and growth plate.