目的 观察不同年龄SD大鼠膝关节对热损伤的耐受能力,探究年龄因素诱导膝关节骨关节炎发生的机制.方法 雄性SD大鼠按月龄分3组:青年组(1月龄)、中年组(6月龄)、老年组(18月龄).取各年龄组大鼠各3只,大鼠双侧膝关节腔注入100 μL浓度为100 mg/L的Cu9S5@SiO,用980 nm、0.72 W/cm2激光照射右侧膝关节,温度上升至50℃后持续5 min,左侧膝关节作为对照组.4周后,观察大鼠膝关节软骨组织损伤情况.分别提取青、中、老年SD大鼠原代膝关节软骨细胞,检测3种细胞的端粒相对长度鉴定软骨细胞的衰老程度.依据来源将提取的软骨细胞分为青年、中年、老年.CCK-8法检测各年龄软骨细胞正常培养时的增殖能力及对过氧化氢(H2O2)和白细胞介素(IL)-1β的耐受能力.使用50μmol/L H2O2和100 ng/mLIL-1β分别干预3种软骨细胞后以TUNEL凋亡检测试剂盒检测细胞凋亡情况.结果 光热损伤4周后青、中、老年组大鼠软骨损伤程度依次增加.CCK-8法显示青、中、老年大鼠膝关节软骨细胞增殖能力依次减弱(P<0.05).在H2O2作用下,中年大鼠软骨细胞抵抗能力最强,青年次之,老年最弱.IL-1β作用下,当其浓度≤200 ng/mL时能促进青、中年大鼠软骨细胞增殖能力,浓度>60 ng/mL时明显抑制老年大鼠软骨细胞增殖能力.TUNEL细胞凋亡检测显示50 μmol/L H2O2干预后青、中、老年大鼠软骨细胞凋亡比例依次减低(P<0.05);100 ng/mL IL-1β干预后各年龄大鼠软骨细胞凋亡均无明显增高(P>0.05).结论 不同年龄大鼠软骨细胞的增殖能力、对活性氧(ROS)和IL-1β的耐受能力差异显著.年龄越大,其自身增殖活性越低,抵抗外界损伤的能力越弱.初步验证了年龄因素诱导骨关节炎发生的机制.
Objective To observe the thermal tolerance of the knee joints in SD rats of different age,and to explore the development mechanism of age-related osteoarthritis (OA).Methods Male SD rats were divided into 3 groups:young (1-month-old),adult (6-month-old) and old rats (18-month-old).A volume of 100 μL Cu9S5@SiO2 nanoparticles was injected into the both knee joints in all 3 groups.The knee joints in the right side were irradiated with the 980 nm,0.72 W/cm2 laser.The temperature of the joint cavity was maintained for 5 minutes after it rose to 50℃.The knee joints were not irradiated and used as control.The knee joint damage was evaluated after 4 weeks.Primary chondrocytes were isolated from the knee joints.The senescence of chondrocytes was evaluated by means of the length of the telomere.Chondrocytes were divided into 3 groups by their origin:young,adult and old rats.CCK 8 was used to evaluate the proliferative ability of these chondrocytes with or without the addition of H2O2 and IL-1β.The apoptosis of chondrocytes exposed to 50 μmol/L H2O2 and 100 ng/mL IL 1β was assessed by TUNEL.Results At 4 weeks after the thermal injury,the severity of cartilage damage was increased in the following order:young,adult and old rats;while the proliferative ability of the chondrocytes decreased in the same age sequence(P<0.05).The resistance capacity towards H2O2 decreased from the adult,young to the old rats.IL-1β at a concentration below 200 ng/mL exhibited positive effect on the chondrocytes from the young and adult rats,whereas exerted inhibitory effect on the chondrocytes from the old rats when the concentration was over 60 ng/mL.Upon the addition of 50 μmol/L H2O2,the apoptosis ratio was reduced in an order of young,adult and old SD rats(P<0.05),but there was no significant difference among different age group under the exposure to 100 ng/mL IL 1β(P>0.05).Conclusion The proliferative capacity and resistance capacity towards ROS and IL-1β of chondrocytes varied significantly among different age groups.The older the age,the lower the proliferation activity and the weaker the resistance capacity to external injury.The mechanism of agerelated osteoarthritis was preliminarily validated.