光热效应及年龄因素对软骨细胞及骨关节炎模型制备的影响

Effects of photothermal effects and aging on chondrocytes and the preparation of osteoarthritis animal models

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100μL浓度为100 mg/L的Cu9S5@SiO2,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/mL IL-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50μmol/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 100μL Cu9 S5@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 H2 O2 and IL-1β.The apoptosis of chondrocytes exposed to 50μmol/L H2 O2 and 100 ng/mL IL-1βwas 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 H2 O2 decreased from the adult,young to the old rats.IL-1βat a concentration below200 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 50μmol/L H2 O2,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-1βof 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 age-related osteoarthritis was preliminarily validated.