雷帕霉素调控自噬在骨关节炎软骨细胞退变中的机制研究

Research on the mechanism of Rapamycin regulating autophagy in the degenerative process of osteoarthritis chondrocyte

背景:骨关节炎(Osteoarthritis,OA)的发生与软骨细胞自噬活动降低、细胞稳态失衡密切相关。雷帕霉素是一种常用的自噬诱导剂,能上调自噬活动、维持细胞稳态。目的:明确能否通过雷帕霉素提高软骨细胞自噬相关因子的表达,促进软骨细胞生存。方法:收集正常软骨标本5例,中期及晚期OA软骨标本各10例。免疫组织化学检测软骨组织中自噬相关蛋白表达情况。体外分离、培养软骨细胞,进行药物实验。各期OA标本软骨细胞分为低、中、高浓度实验组和对照组,分别予以1μmol/L、5μmol/L、10μmol/L雷帕霉素及相同体积培养基。运用聚合酶链式反应(polymerase chain reaction,PCR)及Western-Blot检测自噬相关因子ULK1、Beclin1、LC3表达变化规律。结果:免疫组织化学检测发现,随着OA退变程度加重,软骨组织中自噬相关蛋白阳性表达率逐渐降低。PCR及Western-Blot发现,雷帕霉素干预正常软骨细胞后,实验组自噬相关因子ULK1、Beclin1、LC3表达量高于对照组,且与雷帕霉素呈浓度依赖性。中期OA软骨细胞结果与正常软骨细胞类似,但组间差异更明显。晚期OA软骨细胞中,实验组自噬相关因子表达量较对照组呈上升趋势,但升高幅度较小。结论:雷帕霉素可以上调自噬相关因子的表达,其中正常及中期OA软骨细胞对雷帕霉素诱导的自噬活动具有良好的反应性,而晚期OA软骨细胞对雷帕霉素刺激反应较差。

Background： Osteoarthritis is the result of chondrocytes degeneration. While autophagy is a cellular homeostasis mechanism that plays an essential role in preventing chondrocytes degeneration. Rapamycin as an autophagy inducer could upregulate autophagy activity and maintain cell homeostasis. Objective： To observe the changes of expression of autophagy related molecules（ULK1, Beclin1 and LC3） after regulation of mTOR signal pathway by Rapamycin. Methods： Five normal cartilage samples and a total of 20 moderate and severe osteoarthritis cartilage samples with 10 of each were collected from amputation or knee arthroplasty surgery. Immunohistochemical techniques were used to detect the expression of autophagy-related proteins such as ULK1, Beclin1 and LC3 in cartilage. Knee cartilage cells were isolated and cultured in vitro for drug experiment. Osteoarthritis chondrocyte samples were divided into four groups： low, medium, high concentration Rapamycin groups and blank control group, treated with 1 μmol/L, 5 μmol/L, 10 μmol/L Rapamycin and the same volume of medium, respectively. PCR and Western-blot tests were performed to detect the m RNA and protein expression of autophagy related molecules（ULK1, Beclin1 and LC3）. Results： The results of immunohistochemistry showed that the expression of ULK1, Beclin1 and LC3 in the cartilage decreased significantly with the degradation of osteoarthritis. The results of PCR and Western-blot tests showed that in the normal chondrocyte, autophagy related molecules（ULK1, Beclin1 and LC3） were significant higher in low, medium and high concentration Rapamycin groups than those in blank control group, and were concentration-dependent on Rapamycin. The results of experiment on moderate osteoarthritis chondrocyte were similar to those of the normal chondrocyte, while the difference among groups was more significant. In the severe osteoarthritis chondrocyte, autophagy related molecules were higher in low, medium and high concentration Rapamycin groups than those in the blank control group, but no significant difference was found. Conclusions： Rapamycin can increase the expression of autophagy related molecules such as ULK1, LC3 and Beclin1. The autophagy activity is enhanced in the normal chondrocyte and the moderate osteoarthritis chondrocyte significantly by Rapamycin, but that in the severe osteoarthritis chondrocyte is little changed by Rapamycin.